Capping of unprotected amino groups during peptide synthesis

ABSTRACT

The present invention relates to a method for the synthesis of a polypeptide comprising a pre-determined amino acid sequence. The method according to the invention comprises coupling cycles of coupling an N-terminally protected amino acid building block C-terminally at an unprotected N-terminal amino group of an amino acid chain, wherein at least one coupling cycle comprises a coupling step (a), a capping step (b), and a de-protecting step (c).

RELATED APPLICATIONS

This application claims the benefit of European Patent Application No.18166551.4, filed Apr. 10, 2018, the entire disclosure of which ishereby incorporated herein by reference in its entirety.

DESCRIPTION

The present invention relates to a method for the synthesis of apolypeptide comprising a pre-determined amino acid sequence. The methodaccording to the invention comprises coupling cycles of coupling anN-terminally protected amino acid building block C-terminally at anunprotected N-terminal amino group of an amino acid chain, wherein atleast one coupling cycle comprises a coupling step (a), a capping step(b), and a de-protecting step (c).

The invention further relates to a composition comprising 0.5-5% v/v ofacetic anhydride and 0.2-2% v/v diisopropylethylamine, as well as itsuse as capping reagent for acetylation of an unprotected amino group inpolypeptide synthesis.

Established methods of solid phase peptide synthesis teach coupling ofthe pre-determined C-terminal amino acid of the amino acid chain to besynthesized to a polymer carrier via a linker. The amino acid used forcoupling is an amino acid building block having an N-terminallyprotected amino group, said protective group being a temporarily linkedFmoc group. After successful coupling, the Fmoc protective group iscleaved and the next Fmoc-protected amino acid building block is coupledwith the free amino function of the previous amino acid building block.When the desired amino acid chain is synthesized, it is cleaved from thesolid phase. FIG. 1 is giving an overview of the described approach.

As the coupling reaction is not always complete, the remaining freeamino groups can be acetylated with acetic anhydride (see FIG. 1). Thisreaction is called capping. The aim of capping is preventing theoccurrence of (N−1) impurities (i.e. products lacking an amino acidbuilding block at one position), which are probably hard to separatefrom the desired end product.

In the literature, numerous capping mixtures for the solid phasesynthesis of peptides are described. Fields et al. (PNAS 85, 1384-1388,1988) uses acetic anhydride and diisopropylethylamine (DIPEA) inmethylene chloride for the synthesis of gramicidine A. First, 1equivalent of DIPEA in 20 ml methylene chloride is added. After fiveminutes, 4 equivalents of acetic anhydride are added. The totalincubation time is 20 minutes. Eritja et al. (Tetrahetron 43, 2675-2680,1987) teaches the use of approximately 9% of acetic anhydride andapproximately 16% of DIPEA in DMF combined with an incubation time of 30minutes for the capping process. Echner et al. discloses a mixture ofacetic anhydride, pyridine and methylene chloride for the cappingprocess (Liebigs Ann. Chem. 1988, 1095-1097).

The solid phase synthesis of lixisenatide (also known as AVE0010 orZP-10) described in WO 01/04156 A1, which is enclosed herein byreference, comprises coupling of the individual Fmoc-protected aminoacid building blocks in each the same way, without any capping reaction.

Lixisenatide has the sequence desPro³⁶exendin-4(1-39)-Lys₆-NH₂. Thissubstance is disclosed in WO 01/04156, SEQ ID NO:93 (cf. SEQ ID NO:1 andFIG. 2 of the present application). Exendins are a group of peptideswhich can lower the blood glucose concentration. Exendins have a certainsimilarity to the sequence of GLP-1(7-36) (53%, Göke et al., J. Biol.Chem. 268, 19650-55). Exendin-3 and exendin-4 stimulate an increasingcellular cAMP production in pancreatic acinar cells of guinea pigs byinteraction with the exendin receptors (Raufman, 1996, Reg. Peptides61:1-18). In contrast to exendin-4, exendin-3 effects an increase ofamylase release in pancreatic acinar cells. Exendins act as GLP-1antagonists.

Glucagon-like peptide 1 (GLP-1) is an endocrine hormone which enhancesthe insulin response after oral uptake of glucose or fat. GLP-1generally lowers the glucagon concentrations, slows down gastricemptying, stimulates the (pro-) insulin biosynthesis, increases thesensibility to insulin and stimulates the insulin-independent glycogenbiosynthesis (Hoist (1999), Curr. Med. Chem. 6:1005, Nauck et al.(1997), Exp. Clin. Endocrinol. Diabetes 105:187, Lopez-Delgado et al.(1998), Endocrinology 139:2811). Human GLP-1 has 37 amino acid residues(Heinrich et al., Endocrinol. 115:2176 (1984), Uttenthal et al., J.Clin. Endocrinol. Metabol. (1985), 61:472). Active fragments of GLP-1include GLP-1(7-36) and GLP-1(7-37).

It was suggested that exendin-3, exendin-4 and exendin agonists can beused for the treatment of diabetes mellitus and the prevention ofhyperglycemia, as they reduce gastric emptying and motility (U.S. Pat.No. 5,424,286 and WO 98/0535 A1).

Exendin analogues may be characterized by amino acid substitutionsand/or C-terminal truncations of the native exendin-4 sequence. Suchexendin analogues are described in WO 99/07404, WO 99/25727 and WO99/25728.

The inventors have found that the crude product of a common solid phasesynthesis of lixisenatide with Fmoc-protected amino acid building blocksexhibits certain acetylated erroneous sequences in increased amountswith regard to other acetylated erroneous sequences. These unexpectedlypronounced impurities are in particular Ac(20-44), Ac(17-44), Ac(13-44),Ac(10-44) and Ac(4-44).

The problem to be solved by the present invention is to reduce theamount of acetylated erroneous sequences occurring in peptide synthesisand therefore, to enhance the yield of the peptide synthesis. Inparticular, the amount of those acetylated erroneous sequences having anincreased amount in the crude product should be reduced.

It has been found that during the capping step of lixisenatide solidphase synthesis, the Fmoc-protective group is undesirably cleaved fromsome of the amino acid building blocks just coupled. This undesirablecleavage was only detected for capping at certain amino acid positionsduring the synthesis cycle. Five of these positions are:

-   -   capping after coupling of Arg(20),    -   capping after coupling of Glu(17),    -   capping after coupling of Gln(13),    -   capping after coupling of Leu(10),    -   capping after coupling of Gly(4).

A further problem to be solved by the present application is thus, toprovide capping conditions under which the undesired acetylated productsare no longer present in such increased amounts.

Surprisingly, it was found that applying milder capping conditions leadsto lower by-product concentrations in the crude product or even theircomplete eradication. By reducing or completely eliminating theby-products Ac(17-44), Ac(13-44), and Ac(10-44), whose chromatographicpeaks are close to that of the intended product, the purification of theintended product from the crude product is strongly facilitated. As lessmixed fractions occur, the yield of purified lixisenatide increases. Theyield further increases as the concentration of by-products Ac(20-44)and Ac(4-44), the peaks of which are further away from the intendedproduct peak, are also minimized by the mild capping conditions.

One aspect of the present invention relates to a method for thesynthesis of a polypeptide comprising a pre-determined amino acidsequence, the method comprising coupling cycles of amino acid buildingblocks to an amino acid chain, wherein said amino acid building blockscomprise an unprotected C-terminal carboxyl group and a protectedN-terminal amino group, and wherein said amino acid chain comprises anunprotected N-terminal amino group, wherein at least one coupling cyclecomprises the steps:

-   -   (a) coupling the amino acid building block C-terminally at the        unprotected N-terminal amino group of the amino acid chain, so        that an amide bond is formed between the amino acid chain and        the amino acid building block,    -   (b) contacting the product obtained in step (a) with a capping        reagent comprising a capping compound, wherein the capping        compound binds to an unprotected N-terminal amino group of the        amino acid chain to which no building block has been coupled in        step (a), and    -   (c) de-protecting the N-terminal amino group of the amino acid        building block.

In the present invention, “capping reagent”, “capping composition” or“capping mixture” are used interchangeably. The reagent may be preparedbefore step (b), or the components of the capping reagent are addedduring step (b).

An amino acid chain, capped in step (b), is not capable of coupling afurther amino acid building block, so that chain elongation of thismolecule is terminated.

During the synthesis of the polypeptide, coupling cycles of amino acidbuilding blocks are performed such that the amino acid chain of thepolypeptide is formed building block per building block. For thecoupling step, state-of-the-art techniques can be used, in particular asolid phase synthesis on the basis of Fmoc-protected amino acid buildingblocks. All kinds of solid phases suitable for the solid phase synthesisof peptides can be used. In particular, a solid phase comprising a resincan be used. The resin can be a Rink resin (Rink amide resin) or aTentagel® resin. In a preferred aspect, the solid phase resin is a Rinkresin or Rink amide resin.

Cycles comprising the steps (a), (b) and (c) according to the inventioncan be repeated once or several times. For each amino acid buildingblock to be coupled one cycle is performed. The respective amino acidbuilding block is independently selected for each cycle dependent on thepre-determined amino acid sequence. All kinds of amino acid buildingblocks, as described herein, can be used.

The method according to the invention can combine coupling cycles whichare the same or different in relation to the capping step conditions,such as the capping temperature, the capping reagent composition or/andthe capping duration. In one aspect all coupling cycles according to theinvention are performed under the same capping conditions. In anotheraspect at least one capping step differs from the other capping stepsaccording to the invention, e.g. by a modified capping temperature, thecapping reagent composition or/and the capping duration. In one aspect,the method according to the invention comprises more than one cappingstep condition, e.g. two, three, four or five different capping stepconditions over the whole polypeptide synthesis.

In one aspect, the method according to the invention comprises at leastone coupling cycle, at least two coupling cycles, at least threecoupling cycles, at least four coupling cycles, or at least fivecoupling cycles comprising a capping step (b).

The capping compound is preferably selected from the group consisting ofacetic anhydride (CAS 108-24-7), homologues of acetic anhydride, benzoylchloride (CAS 98-88-4), N-(benzyloxycarbonyloxy)succinimide (CAS13139-17-8), benzyl chloroformate (CAS 501-53-1), esters of chloroformicacid, 1-acetylimidazole (CAS 2466-76-4), di-tert-butyl dicarbonate (CAS24424-99-5) and N-(tert-butoxycarbonyloxy)succinimide (CAS 13139-12-3).In a preferred aspect, the capping compound is selected from aceticanhydride and homologues of acetic anhydride and is preferably aceticanhydride.

In one aspect, the capping reagent comprises acetic anhydride in aconcentration of 0.5-5% v/v. In a preferred aspect the concentration ofacetic anhydride is 1-3% v/v, more preferred 2% v/v.

In another aspect, the capping reagent comprises homologues of aceticanhydride in a concentration of 0.5-5% v/v. In a preferred aspect theconcentration of the homologues of acetic anhydride is 1-3% v/v, morepreferred 2% v/v.

In another aspect, the capping reagent comprises benzoyl chloride in aconcentration of 0.5-5% v/v. In a preferred aspect the concentration ofbenzoyl chloride is 1-3% v/v, more preferred 2% v/v.

In another aspect, the capping reagent comprisesN-(benzyloxycarbonyloxy)succinimide in a concentration of 0.5-5% v/v. Ina preferred aspect the concentration ofN-(benzyloxycarbonyloxy)succinimide is 1-3% v/v, more preferred 2% v/v.

In another aspect, the capping reagent comprises benzyl chloroformate ina concentration of 0.5-5% v/v. In a preferred aspect the concentrationof benzyl chloroformate is 1-3% v/v, more preferred 2% v/v.

In another aspect, the capping reagent comprises esters of chloroformicacid, in a concentration of 0.5-5% v/v. In a preferred aspect theconcentration of the esters of chloroformic acid is 1-3% v/v, morepreferred 2% v/v.

In another aspect, the capping reagent comprises 1-acetylimidazole, in aconcentration of 0.5-5% v/v. In a preferred aspect the concentration of1-acetylimidazole is 1-3% v/v, more preferred 2% v/v.

In another aspect, the capping reagent comprises esters of di-tert-butyldicarbonate, in a concentration of 0.5-5% v/v. In a preferred aspect theconcentration of di-tert-butyl dicarbonate is 1-3% v/v, more preferred2% v/v.

In another aspect, the capping reagent comprises esters ofN-(tert-butoxycarbonyloxy)succinimide in a concentration of 0.5-5% v/v.In a preferred aspect the concentration ofN-(tert-butoxycarbonyloxy)succinimide is 1-3% v/v, more preferred 2%v/v.

The capping reagent can comprise diisopropylethylamine (also termedDIPEA, N-ethyldiisopropylamine or N,N-diisopropylethylamine). In oneaspect, the capping reagent comprises diisopropylethylamine, wherein theconcentration of diisopropylethylamine can be 0.2-2% v/v and preferablyis 0.5-2% v/v. A preferred concentration of diisopropylethylamine is 1%v/v.

In one aspect, the capping reagent comprises diisopropylethylamine andacetic anhydride, wherein the concentration of diisopropylethylamine canbe 0.2-2% v/v and preferably is 0.5-2% v/v, and the concentration ofacetic anhydride can be 0.5-5% v/v and preferably is 1-3% v/v.

In one aspect, the capping composition or capping reagent comprisesdiisopropylethylamine in a concentration of about 1% v/v, and aceticanhydride in a concentration of about 2% v/v.

In the method of the invention, the N-terminal amino group of the aminoacid building block preferably is a base-labile protecting group, morepreferably Fmoc.

The solvent used in the capping step is preferably a polar non-aqueoussolvent, such as acetonitrile, dimethyl sulfoxide (DMSO), methanol,methylene chloride, N,N-dimethylacetamide (DMA), N,N-dimethylformamide(DMF), N-methylpyrrolidone, or mixtures thereof. In a preferred aspect,the solvent used in the capping step is DMF.

In the present invention, the term “about” or “approximately” means arange of ±10%, ±5% or ±1%.

The capping reaction (b) according to the invention can be performed atroom temperature. Room temperature according to the invention is relatedto a temperature between about 15-25° C., a temperature ranging fromabout 20-23° C., a temperature ranging from about 19-21° C. or atemperature of about 20° C.

In the method of the invention, step (b) preferably is performed for 5to 15 min, preferably for 10 min.

In a preferred aspect, step (b) is performed for 10 min with a cappingreagent comprising 2% v/v acetic anhydride and 1% v/v DIPEA.

In another preferred aspect, step (b) is performed for 10 min with acapping reagent comprising 2% v/v acetic anhydride and 1% v/v DIPEA inDMF at positions Arg(20), Glu(17), Gln(13), Leu(10) or/and Gly(4) of thelixisenatide or exendin-4 sequence.

In yet another preferred aspect, step (b) is performed for 10 min with acapping reagent comprising 2% v/v acetic anhydride and 1% v/v DIPEA inDMF at positions Arg(20), Glu(17), Gln(13), Leu(10) and Gly(4) of thelixisenatide or exendin-4 sequence at room temperature, as describedherein.

In one aspect, the method according to the invention can comprise atleast one coupling cycle, at least two coupling cycles, at least threecoupling cycles, at least four coupling cycles or at least five couplingcycles without a capping step, in particular at positions different fromArg(20), Glu(17), Gln(13), Leu(10) or/and Gly(4) of the lixisenatide orexendin-4 sequence.

In another aspect, the method according to the invention comprisescapping according to step (b) after coupling of the amino acid buildingblock Arg(20), Glu (17), Gln(13), Leu(10) or/and Gly(4) of thelixisenatide or exendin-4 sequence, in particular for about 10 min witha capping reagent comprising 2% v/v acetic anhydride and 1% v/vdiisopropylethylamine. Step (b) can be performed after coupling of anArg, Glu, Gln, Leu or/and Gly residue in step (a), in particularArg(20), Glu (17), Gln(13), Leu(10) or/and Gly(4) of the lixisenatide orexendin-4 sequence. Preferably, in other amino acid positions, nocapping step is performed, or/and capping is performed for 20 min with10% acetic anhydride and 5% v/v DIPEA in DMF. In particular, capping isperformed at room temperature, as described herein.

In yet another aspect of the invention the method according to theinvention comprises capping according to step (b) after coupling of allamino acid building blocks of the lixisenatide or exendin-4 sequence, inparticular for about 10 min with a capping reagent comprising 2% v/vacetic anhydride and 1% v/v diisopropylethylamine. Step (b) can beperformed after coupling of all amino acid residues of the lixisenatideor exendin-4 sequence in step (a). In single amino acid coupling steps,capping can be omitted. Step (b) can be performed after coupling of atleast 30 or at least 35 amino acid residues of the lixisenatide orexendin-4 sequence in step (a).

In one aspect, the method according to the invention can comprise atleast one coupling cycle without a capping step (b) and at least onecoupling cycle comprising a capping step (b) as described herein. In oneaspect, the method according to the invention can comprise at least onecoupling cycle, at least two coupling cycles, at least three couplingcycles, at least four coupling cycles, or at least five coupling cycleswithout a capping step (b) and at least one coupling cycle, at least twocoupling cycles, at least three coupling cycles, at least four couplingcycles, or at least five coupling cycles comprising a capping step (b)as described herein. No capping is performed in particular at positionsdifferent from Arg(20), Glu(17), Gln(13), Leu(10) or/and Gly(4) of thelixisenatide or exendin-4 sequence.

In one aspect, the method according to the invention can comprise atleast one coupling cycle, at least two coupling cycles, at least threecoupling cycles, at least four coupling cycles, or at least fivecoupling cycles comprising the steps (a), (b′) and (c), wherein cappingstep (b′) is performed under different conditions as capping step (b). Acoupling cycle comprising step (b′) is particularly applied for couplingof an amino acid building block, different from Arg(20), Glu(17),Gln(13), Leu(10) or/and Gly(4) of the lixisenatide or exendin-4sequence. In one aspect, step (b′) can use a capping reagent comprisingabout 10% v/v acetic anhydride and about 5% v/v diisopropylethylamine inDMF, e.g. performed for about 20 min.

In one aspect, the method according to the invention can comprise atleast one coupling cycle comprising a capping step (b) according to theinvention and at least one coupling cycle comprising a capping step (b′)as described herein. In one aspect, the method according to theinvention can comprise at least one coupling cycle, at least twocoupling cycles, at least three coupling cycles, at least four couplingcycles, or at least five coupling cycles comprising a capping step (b)according to the invention and at least one coupling cycle, at least twocoupling cycles, at least three coupling cycles, at least four couplingcycles, or at least five coupling cycles comprising a capping step (b′)as described herein.

For coupling of an amino acid building block as described herein,comprising coupling of more than one amino acid in one single cycle, acoupling cycle comprising step (b′) is preferably used, e.g. for thecoupling of dipeptides such as Pro-Pro or His-Gly by using the aminoacid building blocks Fmoc-Pro-Pro-OH and Fmoc-His(Trt)-Gly-OH,respectively.

Methods for the performance of a coupling step according to step (a) anda de-protecting step according to step (c) are known by the personskilled in the art. The peptide synthesis is preferably performed in theform of a solid phase synthesis. In a preferred aspect, the couplingcycles are performed from the C-terminus to the N-terminus of thesequence to be synthesized. Reaction conditions for steps (a) and (c)applied in a solid phase peptide synthesis from the C-terminus to theN-terminus by amino acid building blocks are known by the person skilledin the art.

An amino acid building block according to the invention is a compoundwhich is prolonging the amino acid chain to be synthesized by one ormore amino acids in one cycle of the peptide synthesis. In a preferredaspect, an amino acid building block according to the invention prolongsthe amino acid chain to be synthesized by 1, 2, 3, or 4 amino acids. Ina particularly preferred aspect, the amino acid building block accordingto the invention prolongs the amino acid chain to be synthesized by oneor two amino acids.

The amino acid building block according to the invention preferablycomprises one amino acid (mono amino acid building block) or anoligopeptide comprising 2, 3, 4 or more amino acids. In a preferredaspect, the amino acid building block according to the inventioncomprises one amino acid or a peptide, comprising two amino acids suchas e.g. Pro-Pro or His-Gly. Amino acids of an amino acid building blockcomprising more than one amino acid are preferably linked by peptidebonds. Particularly preferred amino acid building blocks comprising twoamino acids are Fmoc-Pro-Pro-OH and Fmoc-His(Trt)-Gly-OH.

It was found that using Fmoc-His(Trt)-Gly-OH instead of amino acidbuilding blocks for His and Gly at positions 1 and 2 in the synthesis oflixisenatide and exendin-4 enables the prevention of undesiredDesGly(2)-lixisenatide. Moreover, the obtained lixisenatide did not showenhanced values of D-His resulting from racemization.

Fmoc-His(Trt)-Gly-OH can e.g. be formed by a method comprising the stepsof:

i) reacting Fmoc-His(Trt)-OH and H-Gly-OBzl tosylate, andii) cleaving the benzyl group of the product obtained in step i) toobtain Fmoc-His(Trt)-Gly-OH.

Exemplary reaction conditions are set forth in example 3.

The amino acid building blocks according to the invention can comprisesuitable modifications in order to selectively prolong the amino acidchain at the desired positions only. Modifications of the amino acidbuilding block can be performed at the N-terminus, at the C-terminusor/and at the side chains of the amino acids.

To protect the N-terminal amino function of the amino acid buildingblock (i.e. the amino group which is, after successful coupling, theN-terminus of the amino chain), all kinds of protective groups commonlyused for the synthesis of peptides, especially for the solid phasesynthesis of polypeptides, can be used. The person skilled in the artknows those kinds of suitable temporary protective groups. In apreferred aspect, protective groups which are unstable in alkalineenvironment can be used. In a preferred aspect the N-terminal aminogroup of the amino acid building block is protected by an Fmocprotective group.

The C-terminal carboxy group of the amino acid building block preferablyremains unprotected.

The amino acid building block according to the invention can comprise,independently from one another, D-amino acids and glycine, L-amino acidsand glycine or/and combinations thereof. In a preferred aspect the aminoacids of the amino acid building block according to the invention areselected independently from one another from L-amino acids and glycine.In a preferred aspect, the amino acids can be selected from α-aminoacids. In a further aspect the amino acids can be selected fromnaturally occurring amino acids such as amino acids naturally occurringin polypeptides. In another aspect the amino acid building blockaccording to the invention can comprise artificial amino acids such asMet(O) (methionine sulfoxide or methionine sulfone), Trp(O₂)(N-formylkynurenine) or/and isoAsp (β-aspartate or isoaspartate). In astill further preferred aspect the amino acids are selected from Ser,Thr, Trp, Lys, Ala, Asn, Asp, Val, Met, Phe, Ile, Pro, Arg, Glu, Gln,Leu, in particular each in the D-form or each in the L-form, and Gly. Ina particularly preferred aspect the amino acid building block accordingto the invention comprises amino acids selected from Arg, Glu, Gln, Leu,in particular each in the D-form or each in the L-form, and Gly.

In particular the amino acids are selected independently from eachother, for example independently from Ser, Thr, Trp, Lys, Ala, Asn, Asp,Val, Met, Phe, Ile, Pro, Arg, Glu, Gln, Leu, in particular each in theD-form or each in the L-form, and Gly.

In one aspect, at least one side chain of the amino acid building blockaccording to the invention can be protected by a further protectivegroup. The further protective group is preferably orthogonal to theN-terminal protective group. Suitable protective groups for said sidechains are known by the person skilled in the art. Examples for suitableprotective groups are e.g. Trt, Boc, Bzl, Pdf, tBu and OtBu, which canbe used for the protection of specific side chains. The person skilledin the art is aware of which side chain needs to be protected by whichkind of protective group. In one aspect, amino acid building blocks asmentioned in Example 1.4 can be used. In case the amino acid buildingblock comprises more than one side chain, one or more of these sidechains can be protected by protective groups, independently selectedfrom suitable protective groups as known by the person skilled in theart.

The polypeptide to be synthesized may be each possible peptide with apre-determined sequence. In a preferred aspect, the polypeptide to besynthesized is a GLP-1 agonist. The polypeptide can be a GLP-1 agonist,wherein the GLP-1 agonist is selected from the group consisting of GLP-1and analogues and derivatives thereof, exendin-3 and analogues andderivatives thereof, exendin-4 and analogues and derivatives thereof. Ina preferred aspect the polypeptide is selected from the group consistingof exendin-4 and lixisenatide. Lixisenatide is most preferred. In afurther preferred aspect the polypeptide is selected from albiglutide,dulaglutide and semaglutide.

Exendin-3, analogues and derivatives of exendin-3, exendin-4 andanalogues and derivatives of exendin-4 are described in WO 01/04156, WO98/30231, U.S. Pat. No. 5,424,286, EP 99610043.4 and WO 2004/005342.These documents are incorporated herein by reference. Exendin-3,exendin-4 and the analogues and derivatives thereof described in thesedocuments can be synthesized by the method according to the invention,whereas additional modifications can be performed after completion ofthe synthesis.

Lixisenatide (SEQ ID NO:1, FIG. 2), exendin-4 (SEQ ID NO:2, FIG. 2) andexendin-3 (SEQ ID NO:3, FIG. 2) have a high degree of sequence identity.Sequences of lixisenatide and exendin-4 are identical at positions 1-37.Sequence 1-39 of exendin-4 is identical to exendin-3 at 37 of 39positions (94%) (J. Biol. Chem. 267, 1992, 7402-7405). Sequencepositions are given herein with respect to the sequence of lixisenatideor exendin-4. Starting from these sequences, the person skilled in theart can readily determine corresponding positions in other sequences.

Analogues and derivatives of exendin-3 or/and exendin-4 particularlycomprise a modified amino acid sequence. In one aspect, the amino acidsequence is modified by deletion of one or more amino acids (e.g.desPro³⁶, desPro³⁷, desAsp²⁸, desMet(O¹⁴) in exendin-4 and therespective positions in exendin-3). In one aspect, one or more aminoacids can be replaced (e.g. Met(O¹⁴), Trp(O₂)²⁵, isoAsp²⁸, Asp²⁸, Pro³⁸in exendin-4 and the respective positions in exendin-3), whereinnaturally occurring or artificial amino acids such as e.g. Met(O)(methionine sulfoxide or methionine sulfone), Trp(O₂)(N-formylkynurenine) or/and isoAsp (β-aspartate or isoaspartate) can beintroduced. Artificial amino acids can readily be introduced in thesequence by using the respective amino acid building blocks in thesynthesis cycle.

In one aspect the C-terminus or/and the N-terminus of the polypeptidecan be modified, e.g. by the addition of sequences such as -(Lys)-,-(Lys)₂-, -(Lys)₃-, -(Lys)₄-, -(Lys)₅-, -(Lys)₆-, and -Asn-(Glu)₅-. In apreferred aspect, additional amino acid sequences are e.g. -(Lys)₄-,-(Lys)₅-, -(Lys)₆- and -Asn-(Glu)₅-. The C-terminal carboxy group ispreferably an acid amine group (—NH₂). Optionally, the modification ofthe C-terminus or/and the N-terminus is performed in a separate stepafter completion of the synthesis cycles of the method according to theinvention.

After completion of the synthesis cycles of the method according to theinvention, pharmaceutically acceptable salts of the synthesizedpolypeptides can optionally be formed in an additional step. Methods forthe formation of pharmaceutically acceptable salts of polypeptides areknown by the person skilled in the art. Preferred pharmaceuticallyacceptable salts are e.g. acetate salts.

In one aspect, the GLP-1 agonist is preferably selected from the groupconsisting of exendin-4, analogues and derivatives thereof andpharmaceutically acceptable salts thereof.

A further preferred GLP-1 agonist is an analogue of exendin-4 selectedfrom the group consisting of:

H-desPro³⁶-exendin-4-Lys₆-NH₂,H-des(Pro^(36,37))-exendin-4-Lys₄-NH₂,H-des(Pro^(36,37))-exendin-4-Lys₅-NH₂, and pharmaceutically acceptablesalts thereof.

A further preferred GLP-1 agonist is an analogue of exendin-4 selectedfrom the group consisting of

desPro³⁶[Asp²⁸]exendin-4 (1-39),desPro³⁶[IsoAsp²⁸]exendin-4 (1-39),desPro³⁶[Met(O)¹⁴,Asp²⁸]exendin-4 (1-39),desPro³⁶[Met(O)¹⁴,IsoAsp²⁸]exendin-4 (1-39),desPro³⁶[Trp(O₂)²⁶,Asp²⁸]exendin-2 (1-39),desPro³⁶[Trp(O₂)²⁶,IsoAsp²⁸]exendin-2 (1-39),desPro³⁶[Met(O)¹⁴Trp(O₂)²⁶,Asp²⁸]exendin-4 (1-39),desPro³⁶[Met(O)¹⁴Trp(O₂)²⁶,IsoAsp²⁸]exendin-4(1-39), andpharmaceutically acceptable salts thereof.

A further preferred GLP-1 agonist is an analogue of exendin-4 selectedfrom the groups as described above, further modified with a -Lys₆-NH₂peptide at the C-terminus.

A further preferred GLP-1 agonist is an analogue of exendin-4 selectedfrom the group consisting of:

H-(Lys)₆-desPro³⁶[Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,desAsp²⁸Pro³⁶,Pro³⁷,Pro³⁸exendin-4(1-39)-NH₂,H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-desPro³⁶[Trp(O₂)²⁶,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,H-desAsp²⁸ Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁶]exendin-4(1-39)-NH₂,H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁶,Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁶,Asp²⁸]exendin-4(1-39)-NH₂,desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁶,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁶,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-desPro³⁶[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,desMet(O)¹⁴ Asp²⁸ Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-NH₂,H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸] exendin-4(1-39)-NH₂,desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-desPro³⁶[Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,desAsp²⁸Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴, Trp(02)²⁵]exendin-4(1-39)-NH₂,H-(Lys)₆-desPro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,desPro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴,Trp(02)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,and pharmaceutically acceptable salts thereof.

In a further aspect a preferred GLP-1 agonist is selected from the groupconsisting of GLP-1 (in particular GLP-1(7-36) amide, SEQ ID NO:4),Arg³⁴,Lys²⁶(N^(ε)(γ-glutamyl(N^(α)hexadecanoyl))) GLP-1 (7-37)(liraglutide), albiglutide, dulaglutide, semaglutide andpharmaceutically acceptable salts thereof. In particular, a preferredGLP-1 agonist is selected form the group consisting of albiglutide,dulaglutide, semaglutide and pharmaceutically acceptable salts thereof.

A further preferred GLP-1 agonist is lixisenatide (SEQ ID NO:1) as wellas its pharmaceutically acceptable salts.

In one aspect the polypeptide to be synthesized is preferablylixisenatide or exendin-4, wherein, after coupling of the amino acidbuilding block Arg(20), Glu (17), Gln(13), Leu(10) or/and Gly(4), step(b) is performed for about 10 min with a capping reagent comprising 2%v/v acetic anhydride and 1% v/v diisopropylethylamine.

In one aspect the method according to the invention comprises thesynthesis of a polypeptide in form of a solid phase synthesis. Themethod according to the invention optionally comprises a further stepof:

(d) cleaving the polypeptide linked to the solid phase.

Step (d) is particularly performed when the synthesis of the amino acidchain is completed. Step (d) can be performed by cleavage of a solidphase-bound polypeptide from the solid phase, comprising contacting thesolid phase, to which the polypeptide is bound, with a compositionconsisting essentially of trifluoroacetic acid and 1,2-ethanedithiol, ata temperature in the range of about 23° C. to about 29° C.

Cleavage can be performed with a composition comprising trifluoroaceticacid in an amount of about 95 to about 99% v/v.

Cleavage can also be performed with a composition comprising1,2-ethanedithiol in an amount of about 1 to about 5% v/v.

Preferably, cleavage is performed with a composition essentiallyconsisting of trifluoroacetic acid in an amount of about 97% v/v, and1,2-ethanedithiol in an amount of about 3% v/v.

Cleavage can be performed by contacting the composition with the solidphase to which the polypeptide is bound, at a temperature of about 25°C. to about 27° C.,

Preferably, cleavage is performed by contacting the composition with thesolid phase to which the polypeptide is bound, at a temperature of about26° C.

Most preferably, cleavage is performed with a composition essentiallyconsisting of trifluoroacetic acid in an amount of about 97% v/v, and1,2-ethanedithiol in an amount of about 3% v/v, at a temperature ofabout 26° C.

Most preferably, cleavage is performed with a composition essentiallyconsisting of trifluoroacetic acid in an amount of about 97% v/v, and1,2-ethanedithiol in an amount of about 3% v/v, at a temperature ofabout 26° C. for about 4 h.

A preferred aspect of the invention relates to a method for the solidphase synthesis of a polypeptide comprising a pre-determined amino acidsequence, the method comprising coupling cycles of amino acid buildingblocks to an amino acid chain,

wherein said amino acid building blocks comprise an unprotectedC-terminal carboxyl group and a protected N-terminal amino group,and wherein said amino acid chain comprises an unprotected N-terminalamino group,wherein at least one coupling cycle comprises the steps:

-   (a) coupling the amino acid building block C-terminally at the    unprotected N-terminal amino group of the amino acid chain, so that    an amide bond is formed between the amino acid chain and the amino    acid building block,-   (b) contacting the product obtained in step (a) with a capping    reagent comprising acetic anhydride, wherein the acetic anhydride    binds to an unprotected N-terminal amino group of the amino acid    chain to which no building block has been coupled in step (a), and-   (c) de-protecting the N-terminal amino group of the amino acid    building block, wherein the capping reagent comprises 0.5-5% v/v of    acetic anhydride, and 0.2-2% v/v of diisopropylethylamine.

A further aspect of the present invention relates to a compositioncomprising 0.5-5% v/v of acetic anhydride and 0.2-2% v/v ofdiisopropylethylamine in DMF. In a preferred aspect the compositionaccording to the invention comprises 1-3% v/v of acetic anhydride and0-5-2% v/v diisopropylethylamine in DMF, preferably about 2% v/v aceticanhydride and about 1% v/v diisopropylethylamine in DMF.

Yet another aspect of the present invention relates to a compositioncomprising diisopropylethylamine in a concentration of about 1% v/v, andacetic anhydride in a concentration of about 2% v/v.

Yet another aspect of the invention relates to a composition comprisinga homologue of acetic anhydride in a concentration of 0.5-5% v/v. In apreferred aspect the concentration of the acetic anhydride homologue is1-3% v/v, more preferably about 2% v/v. The composition can compriseDIPEA, as described herein.

Yet another aspect of the invention relates to a composition comprisingbenzoyl chloride in a concentration of 0.5-5% v/v. In a preferred aspectthe concentration of benzoyl chloride is 1-3% v/v, more preferably about2% v/v. The composition can comprise DIPEA, as described herein.

Yet another aspect of the invention relates to a composition comprisingN-(benzyloxycarbonyloxy)succinimide in a concentration of 0.5-5% v/v. Ina preferred aspect the concentration ofN-(benzyloxycarbonyloxy)succinimide is 1-3% v/v, more preferably about2% v/v. The composition can comprise DIPEA, as described herein.

Yet another aspect of the invention relates to a composition comprisingbenzyl chloroformate in a concentration of 0.5-5% v/v. In a preferredaspect the concentration of benzyl chloroformate is 1-3% v/v, morepreferably about 2% v/v. The composition can comprise DI PEA, asdescribed herein.

Yet another aspect of the invention relates to a composition comprisingan ester of chloroformic acid in a concentration of 0.5-5% v/v. In apreferred aspect the concentration of the ester of chloroformic acid is1-3% v/v, more preferably about 2% v/v. The composition can compriseDIPEA, as described herein.

Yet another aspect of the invention relates to a composition comprising1-acetylimidazole in a concentration of 0.5-5% v/v. In a preferredaspect the concentration of 1-acetylimidazole is 1-3% v/v, morepreferably about 2% v/v. The composition can comprise DIPEA, asdescribed herein.

Yet another aspect of the invention relates to a composition comprisingdi-tert-butyl dicarbonate in a concentration of 0.5-5% v/v. In apreferred aspect the concentration of di-tert-butyl dicarbonate is 1-3%v/v, more preferably about 2% v/v. The composition can comprise DIPEA,as described herein.

Yet another aspect of the invention relates to a capping compositioncomprising N-(tert-butoxycarbonyloxy)succinimide in a concentration of0.5-5% v/v. In a preferred aspect the concentration ofN-(tert-butoxycarbonyloxy)succinimide is 1-3% v/v, more preferably about2% v/v. The composition can comprise DIPEA, as described herein.

The composition according to the invention can be used for capping offree amino groups in the synthesis of a polypeptide, in particular inthe synthesis of a polypeptide as described herein. Moreover, thecomposition according to the invention can be used for the acetylationof a free carbon-bound amino group as described herein.

In a preferred aspect the composition according to the invention isapplied in step (b) of the method according to the invention, e.g. for aperiod of 10 min, after coupling step (a) of positions Arg(20), Glu(17),Gln(13), Leu(10) or/and Gly(4) of lixisenatide, exendin-3 or exendin-4,or the respective positions of further GLP-1 analogue.

Yet another aspect of the invention is the use of the composition, asdescribed herein, for acetylation of an unprotected amino group inpolypeptide synthesis.

Abbreviations

-   Ac(N1-N2): N-terminally acetylated fragment of a polypeptide from    position N1 to N2.-   H(N1-N2) or (N1-N2): Fragment of a polypeptide from position N1 to    N2 comprising a free, N-terminal amino function.-   Fmoc(N1-N2): Fragment of a polypeptide from position N1 to N2    comprising a protected N-terminal amino function, wherein the    protective group is Fmoc.-   (N−1)-impurity: Relates to the occurrence of an unintended peptide    during peptide synthesis, which lacks a building block at a certain    position. In case the intended synthesized polypeptide has a length    N, the impurity has a length of N−1. The occurrence of (N−1)    impurities is prevented by capping.-   Fmoc fluorenylmethoxycarbonyl-   Boc tert-butoxycarbonyl-   Bzl benzyl-   Pbf 2,2,5,7,8-pentamethyldihydrobenzofuran-5-sulfonyl-   tBu tert-butyl-   OtBu O-tert-butyl-   Trt trityl-   DIPE diisopropylether

The invention is further characterized by the following Figures andExamples.

FIGURES

FIG. 1: Solid phase synthesis of peptides.

FIG. 2: Sequence of lixisenatide (SEQ ID NO:1), exendin-4 (SEQ ID NO:2),exendin-3 (SEQ ID NO:3) and GLP-1 (GLP-1(7-36) amide, SEQ ID NO:4).

FIG. 3: Occurrence of acetylated erroneous sequences during synthesis oflixisenatide. Coupling of Fmoc-Arg(20)-OH and subsequent capping/Fmoccleavage. It should be noted that the position 21 (Leu) was omitted fromthe synthesis. (1) Fmoc-(22-44)+Arg, (2) (22-44)+Arg, (3) Ac(22-44)+Arg,(4) Fmoc-(22-44)+Arg+Val. The data show that the acetylated fragmentshave already been formed during the capping step, however, the wrongposition is acetylated [Ac(22-24)+Arg is already occurring duringcapping of Arg].

FIG. 4: Occurrence of acetylated erroneous sequences during synthesis oflixisenatide. Coupling of Fmoc-Gln(13)-OH and subsequent capping/Fmoccleavage. (1) Ac(14-44), (2) Fmoc(13-44), (3) Ac(13-44), (4) (13-44),(5) (14-44). The data show that the acetylated fragments have alreadybeen formed during the capping step, however, the wrong position isacetylated (Ac(13-44)).

FIG. 5: Occurrence of acetylated erroneous sequences during synthesis oflixisenatide. Coupling of Fmoc-Lys(12)-OH and subsequent capping/Fmoccleavage. (1) Ac(13-44), (2) Fmoc(12-44), (3) Ac(12-44), (4) (12-44).The data show that the acetylated fragments have already been formedduring the capping step, however, the wrong position is acetylated(Ac(12-44)).

FIGS. 6A-6C: Comparison of the synthesis of lixisenatide using themethod of capping according to the invention (FIG. 6B) in comparison tocapping with 10% acetic anhydride and 5% v/v DI PEA in DMF for 20 min(FIG. 6A) by means of HPLC chromatography. (FIG. 6C) overlap of HPLCchromatograms of (FIG. 6A) and (FIG. 6B).

FIG. 7: HPLC of lixisenatide (raw product). Red: undesired acetylatedby-products.

FIG. 8: Ac(36-44) formation, depending upon the capping cocktail andtemperature.

FIG. 9: Ac(23-44) formation, depending upon the capping cocktail andtemperature.

FIG. 10: Ac(21-44) formation, depending upon the capping cocktail andtemperature.

FIG. 11: Ac(19-44) formation, depending upon the capping cocktail andtemperature.

FIG. 12: Ac(18-44) formation, depending upon the capping cocktail andtemperature.

FIG. 13: Ac(15-44) formation, depending upon the capping cocktail andtemperature.

FIG. 14: Ac(12-44) formation, depending upon the capping cocktail andtemperature.

FIG. 15: Ac(8-44) formation, depending upon the capping cocktail andtemperature.

FIG. 16: Ac(6-44) formation, depending upon the capping cocktail andtemperature.

FIG. 17: Comparison of Ac(X-44) content in capping at 9 differentpositions in the lixisenatide synthesis at 15° C., room temperature (RT)and 30° C.

FIG. 18: Comparison of Ac[(X-1)-44] content in capping at 9 differentpositions in the lixisenatide synthesis at 15° C., room temperature (RT)and 30° C.

FIG. 19: Comparison of Ac(X-44) content in capping under differentconditions, or without capping, at 9 different positions in thelixisenatide synthesis under different capping conditions.

FIG. 20: Comparison of Ac[(X-1)-44] content in capping under differentconditions, or without capping, at 9 different positions in thelixisenatide synthesis under different capping conditions.

EXAMPLE 1

Synthesis of Lixisenatide

The active substance Lixisenatide is a polypeptide amide composed of 44amino acids; acetate functions as counterion.

In the one-letter code, the amino acid sequence of Lixisenatide is asfollows:

H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P-P-S-K-K-K-K-K-K-NH₂

The peptide chain was constructed by means of linear solid-phasesynthesis, starting from the C-terminus, Lys-44.

The method of synthesis is Fmoc solid-phase peptide synthesis, in whicha Rink amide resin was used in order to obtain a peptide amide. Thereactions were carried out in DMF at room temperature. Between thereactions, washing was carried out repeatedly, mostly with DMF, with oneof the middle washing steps being carried out with isopropanol.

The synthesis of Lixisenatide on the polymeric support can be brokendown into the following steps:

-   -   Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink        resin    -   Capping of the unreacted amino group    -   Cleavage of the temporary protecting group Fmoc    -   Coupling of the further Fmoc-amino acids or Fmoc-dipeptides    -   Capping of the unreacted amino group    -   Final Fmoc cleavage    -   Cleavage of Lixisenatide from the resin and simultaneous removal        of the side chain protecting groups

The synthesis cycle is illustrated in FIG. 1.

1.1 Coupling of the First Fmoc-Amino Acid (Fmoc-Lys(Boc)-OH) to RinkResin

Before the synthesis began, the Rink amide resin was swollen in DMF. Theswelling was carried out for 2-15 h. Subsequently, the temporaryprotecting group Fmoc was cleaved from the Rink amide resin using 25%piperidine in DMF. This cleavage was undertaken twice; cleavage time of5 minutes and 20 minutes. Following the Fmoc cleavage, the resin waswashed repeatedly with DMF and once with isopropanol.

The coupling of the first Fmoc-amino acid, Fmoc-Lys(Boc)-OH, was carriedout in an excess of 2.4 eq, in order to load the resin. HOBt hydrate,HBTU and DIPEA served as coupling reagents. The coupling time was 60-120min.

In order to completely load the Rink resin with Fmoc-Lys(Boc)-OH, afurther loading was carried out with the coupling reagents HOBt hydrateand DIC. The coupling time was 6-18 h. The mixture was stirred whilestep 1.1 was carried out. The capping was subsequently carried out.

1.2 Capping of the Unreacted Amino Group

The consequence of incomplete loading of the resin is that as yetunreacted amino groups are found on the resin. These were inactivated,and hence made unavailable for further coupling, by adding a mixture ofacetic anhydride/DIPEA/DMF (10:5:85). The capping mixture remained onthe resin for 20 minutes while stirring. The remaining free amino groupis acylated. Subsequently, the resin was washed repeatedly with DMF andonce with isopropanol.

A capping method according to the invention at least at 5 positions of aLixisenatide synthesis is described in examples 4 and 5.

1.3. Cleavage of the Temporary Protecting Group Fmoc

The temporary protecting group Fmoc was cleaved using 25% piperidine inDMF. This cleavage was undertaken twice; cleavage time of 5 minutes and20 minutes. Following the Fmoc cleavage, the resin was washed repeatedlywith DMF and once with isopropanol.

1.4 Coupling of the Further Fmoc-Amino Acids or Fmoc-Dipeptides

The next Fmoc-amino acid was coupled to the deprotected amino group onthe resin. The coupling was carried out in DMF at different equivalents.The coupling times were between 2 h and 18 h. HOBt/DIC, and alsoHBTU/DIPEA, were used as coupling reagents.

The following derivatives were used as Fmoc-amino acids:

-   -   Fmoc-Lys(Boc)-OH    -   Fmoc-Ser(tBu)-OH    -   Fmoc-Pro-OH    -   Fmoc-Ala-OH×H₂O    -   Fmoc-Gly-OH    -   Fmoc-Asn(Trt)-OH    -   Fmoc-Leu-OH    -   Fmoc-Trp(Boc)-OH    -   Fmoc-Glu(OtBu)-OH×H₂O    -   Fmoc-Ile-OH    -   Fmoc-Phe-OH    -   Fmoc-Arg(Pbf)-OH    -   Fmoc-Val-OH    -   Fmoc-Met-OH    -   Fmoc-Gln(Trt)-OH    -   Fmoc-Asp(OtBu)-OH    -   Fmoc-Thr(tBu)-OH    -   Fmoc-His(Trt)-OH

Alternatively, it was also possible to use Fmoc-dipeptides (methodaccording to the invention):

-   -   Fmoc-Pro-Pro-OH (CAS 129223-22-9)    -   Fmoc-Ala-Pro-OH (CAS 186023-44-9)    -   Fmoc-Ser(tBu)-Gly-OH (CAS 113247-80-6)    -   Fmoc-Gly-Pro-OH (CAS 212651-48-4)    -   Fmoc-Gly-Gly-OH (CAS 35665-38-4)    -   Fmoc-Asn(Trt)-Gly-OH (from Bachem B-3630)    -   Fmoc-Glu(OtBu)-Gly-OH (CAS 866044-63-5)    -   Fmoc-His(Trt)-Gly-OH

If the coupling was found to be incomplete according to the Kaiser test(E. Kaiser et al, Anal. Biochem. 34, 1970, 595), further coupling waspossible. For this purpose, the Fmoc-amino acid was coupled again,together with HBTU/DIPEA/HOBt hydrate.

1.5 Capping of the Unreacted Amino Group

See description under point 1.2.

1.6 Final Fmoc Cleavage

The final Fmoc cleavage was carried out as described under point 1.3.The resin was finally washed again with diisopropyl ether and driedunder reduced pressure.

1.7 Cleavage of Lixisenatide from the Resin and Simultaneous Removal ofthe Side Chain Protecting Groups

The cleavage of Lixisenatide from the Rink resin was carried out asdescribed in example 6.

1.8 Synthesis of Lixisenatide with Inventive Use of Dipeptides

The coupling of the first Fmoc-Lys(Boc)-OH to the resin was carried outwith HBTU/DIPEA/HOBt hydrate. After the coupling of the first amino acidFmoc-Lys(Boc)-OH to the free amine of the Rink amide resin, thefollowing process steps were conducted in an endlessly repeating cycle(see also steps 1.3 to 1.6):

-   -   Fmoc cleavage    -   Coupling    -   Further coupling, if necessary    -   Capping    -   After coupling of the final amino acid unit, the N-terminal Fmoc        group is cleaved.

Standard Fmoc-protected amino acids were coupled with DIC/HOBt, with theexcess of amino acids and coupling reagents being between 2 and 4equivalents.

At the positions Pro(36) and Pro(37), instead of two Fmoc-Pro-OH aminoacid derivatives, the dipeptide Fmoc-Pro-Pro-OH was coupled withHBTU/DIPEA.

At the position Pro(31), coupling was carried out with HBTU/DIPEA/HOBthydrate.

At the positions His(1) and Gly(2), instead of the amino acidderivatives Fmoc-His(Boc)-OH and Fmoc-Gly-OH, the dipeptideFmoc-His(Trt)-Gly-OH was coupled.

After the couplings, the capping was carried out in each case withAc₂O/DIPEA, as is described in examples 4 and 5.

The Fmoc cleavage was performed with 25% piperidine in DMF, in each casesuccessively first with 5 minutes of reaction time, then with 20-40minutes of reaction time.

The completeness of the coupling was checked by means of a Kaiser test.

After the last coupling and last cleavage of the Fmoc group, the resinwas washed, firstly repeatedly with DMF, then with isopropanol andfinally with diisopropyl ether, and it was subsequently dried at 35° C.under reduced pressure.

The cleavage of the raw peptide from the resin was carried out intrifluoroacetic acid with scavengers such as 1,2-ethanedithiol.

The raw peptide was purified in a two-step HPLC process with C18 RPsilica gel as solid phase. In the first purification step, a buffersystem with acetonitrile/water with 0.1% TFA was used; in the secondstep, a buffer system with acetonitrile/water with AcOH was used. Afterconcentration of the pooled solutions, the pure peptide was obtained byfreeze-drying.

Use of 3500 g of Rink amide resin with a loading of 0.3 mmol/g (i.e. a1.05 mol batch) gave 9970 g of peptide on resin. 4636 g of raw peptidewere obtained therefrom.

After purification, 576 g of pure peptide were obtained therefrom. MS:4855.5 (monoisotopic molar mass); found 4855.6. Amino acid sequencing:correct sequence found. Assay: 89.0% (as is).

1.9 Synthesis of Lixisenatide without Use of Dipeptides

The peptide chain was constructed by means of linear solid-phasesynthesis, starting from the C-terminus, Lys-44.

Standard Fmoc-protected amino acids were coupled with DIC/HOBt, with theexcess of amino acids and coupling reagents being between 2 and 4equivalents.

At the positions Pro(37), Pro(36), Pro(31), coupling was carried outwith HBTU/DIPEA/HOBt hydrate.

Each coupling was followed by capping with Ac₂O/DIPEA. The Fmoc cleavagewas performed with 25% piperidine in DMF, in each case successivelyfirst with 5 minutes of reaction time, then with 20 minutes of reactiontime.

The completeness of the coupling was checked by means of a Kaiser test.After the last coupling and last cleavage of the Fmoc group, the resinwas washed, firstly repeatedly with DMF, then with isopropanol andfinally with diisopropyl ether, and it was subsequently dried at 35° C.under reduced pressure.

The cleavage of the raw peptide from the resin was carried out intrifluoroacetic acid with scavengers such as 1,2-ethanedithiol,thioanisole, phenol and water.

The raw peptide was purified in a two-step HPLC process with C18 RPsilica gel as solid phase. After concentration of the pooled solutions,the pure peptide was obtained by freeze-drying. Table 1 compares thecontents of racemized D-His-Lixisenatide and the contents of someimpurities in the pure peptide between the synthesis using thedipeptides and without the dipeptides.

TABLE 1 Comparison of the Lixisenatide syntheses with and without use ofthe dipeptides. Content of Content of Content of Content desGly(2)-desPro(36)- diPro(36)- of D-His Lixisenatide Lixisenatide LixisenatideSynthesis of 0.41% Not present Not present Not present lixisenatide withdipeptides Fmoc- His(Trt)-Gly-OH, Fmoc-Pro-Pro- OH according to theinvention Comparative  4.1% 2.5% 1% 1% synthesis of lixisenatide withoutdipeptides

The data show that the use of the dipeptide Fmoc-His(Trt)-Gly-OH gives aLixisenatide which does not contain elevated values of D-His arisingfrom racemization. Moreover, when using Fmoc-His(Trt)-Gly-OH,desGly(2)-Lixisenatide is no longer found. Furthermore, the N−1 and N+1peptides in the vicinity of the chain position Pro(36) and Pro(37) (e.g.desPro(36)-Lixisenatide or diPro(36)Lixisenatide) did not occur.

EXAMPLE 2 Synthesis, Purification and Characterization of Exendin-4(According to the Invention)

The active substance Exendin-4 is a polypeptide amide composed of 39amino acids; acetate functions as counterion.

In the one-letter code, the amino acid sequence is as follows:

H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P-P-P-S-NH₂

MW 4186.66 g/mol; MW (monoisotopic)=4184.03 g/mol.

The synthesis of Exendin-4 was carried out precisely as described in thesynthesis of Lixisenatide, according to the abovementioned sequence. Atpositions 1 and 2, coupling was carried out in one cycle withFmoc-His(Trt)-Gly-OH. At positions 37 and 38, coupling was carried outin one cycle with Fmoc-Pro-Pro-OH. At the other positions, coupling wascarried out with Fmoc-amino acids (monoamino acid units).

Use of 26.666 g of Rink amide resin with a loading of 0.42 mmol/g (i.e.a 11.2 mmol batch) gave 74 g of peptide on resin. From this, 65 g ofpeptide on resin were cleaved, and 28 g of raw peptide were obtained.For the purification, from this, 21.3 g of raw peptide were used, and4.01 g of pure peptide were obtained. MS: 4184.03 (monoisotopic molarmass): found 4185.1 [M+H]. Purity 98.25 FI %.

The use of the dipeptides confirms the results which were obtained forLixisenatide. The use of the dipeptide Fmoc-His(Trt)-Gly-OH gives anExendin-4 which does not contain elevated values of D-His arising fromracemization. Moreover, when using Fmoc-His(Trt)-Gly-OH,desGly(2)-Exendin-4 is no longer found. Furthermore, the N−1 and N+1peptides in the vicinity of the chain position Pro(36) and Pro(37) (e.g.desPro(36)-Exendin-4 or diPro(36)Exendin-4) did not occur.

EXAMPLE 3 Synthesis of Fmoc-his(Trt)-Gly-OH 3.1 Fmoc-his(Trt)-Gly-OBzl

40 g of Fmoc-His(Trt)-OH were dissolved together with 32.7 g ofH-Gly-OBzl tosylate and 29.37 g of HBTU in 400 ml of ethyl acetate.Thereafter, 33.32 ml of N-ethylmorpholine were added. The reaction wasstirred for 4 h at 30° C. Thereafter, extraction was carried out threetimes with 256 g of an 8% sodium bicarbonate solution each time, andthen washing was carried out once with 250 ml of water. Half of theresulting ethyl acetate solution was evaporated and processed further inthe next step.

3.2 Fmoc-his(Trt)-Gly-OH

THF and methanol were added to the ethyl acetate phase, such that a5:2:2 (w/w/w) THF/ethyl acetate/MeOH mixture was formed. Subsequently,10 g of palladium on carbon catalyst (5%) were added, and this mixturewas hydrogenated at 30° C. and a hydrogen pressure of 1.1 bar for 2.5 h.Thereafter, the catalyst was filtered off and the resulting solution wasevaporated until a precipitate began to form. Subsequent stirring wascarried out for 1 h and the solution was left to stand at roomtemperature for 4 days. The product was filtered off and subsequentlyextracted by stirring in 2-butanone at 80° C. for 4 h. Yield: 32.9 g ofFmoc-His(Trt)-Gly-OH (75%).

EXAMPLE 4 Acetylated Erroneous Sequences During the Synthesis ofLixisenatide 4.1 Determining the Content of Acetylated ErroneousSequences During the Synthesis of Lixisenatide

Some acetylated erroneous sequences can be seen in the HPLC profile ofthe crude Lixisenatide product. These usually arise from unreacted aminogroups on the resin being capped. What is achieved by the capping isthat no (N−1) impurities can occur, which differ only slightly from thedesired product and are hence difficult to remove by purification.

The completeness and also the coupling kinetics at selected positionswere monitored by Edman degradation. A resin sample was taken from thesynthesis of Lixisenatide and the Fmoc group was cleaved therefrom. Thisresin sample was then subjected to Edman degradation and in this way itwas possible to determine the ratio of coupled amino acid to the (N−1)amino acid, from which the coupling yield could be directly inferred.The results of the Edman degradation (table 2) show high couplingvalues. These values are so high that they cannot account for theamounts of acetylated erroneous sequences (HPLC data in table 2). Thismeans that there must be an alternative way of forming theseby-products. The elucidation of this situation will be described in thefollowing sections.

TABLE 2 Coupling yields and contents of acetylated fragments duringsynthesis of Lixisenatide. The percentage contents of acetylatederroneous sequences from HPLC data and Edman results (coelution ofAc(6-44), Ac(5-44) and Ac(4-44)) are compared to one another. Amino acidto be Coupling yield Impurities content Impurity coupled (Edman data)(HPLC) Ac(36-44) Ala(35) 99.4-99.5% 4.7% Ac(23-44) Phe(22) >98.4% 0.9%Ac(20-44) Val(19)  99.7% 2.0% Ac(13-44) Lys(12) 98.7-99.5% 2.1% Ac(6-44)Thr(5) 98.4-99.5% Approx. 4.3% Ac(5-44) Gly(4) 99.1-99.8% Ac(4-44)Glu(3) 98.2-99.4%

4.2 Formation of Acetylated Erroneous Sequences

In order to investigate the points in the synthesis cycle at which theacetylated erroneous sequences are formed, resin samples were taken overa coupling cycle, and the peptide was cleaved and investigated usingLC-MS. These investigations were carried out at the positions ofcoupling of Fmoc-Arg(20)-OH and coupling of Fmoc-Gln(13)-OH.

In the coupling of Fmoc-Arg(20)-OH to the solid-phase-bonded peptide ofthe Lixisenatide partial sequence H(22-24), samples were taken aftercoupling times of 1 h, 2 h, 4 h, 8 h and 24 h and also after capping,the subsequent Fmoc cleavage and the coupling of valine(19). As can beseen in FIG. 3, the erroneous sequence Ac(22-44)+Arg occurred for thefirst time during the capping step (3.1%). During the capping,therefore, a small portion of the Fmoc group was cleaved (lost) andimmediately acylated. In order to explain the designation Ac(22-24)+Arg,it should be noted that the position 21 (Leu) was omitted from thesynthesis.

The same experiment was conducted for the coupling of Fmoc-Gln(13)-OHduring the Lixisenatide synthesis (FIG. 4). In this case, the erroneoussequence Ac(13-44) was observed (4.6%) for the first time during theFmoc cleavage after the coupling and the capping of glutamine(13). Inthe remaining course of the synthesis after the coupling ofFmoc-Lys(12)-OH, it can be seen that Ac(12-44) was also formed (4.1%)during the capping (see FIG. 5).

The experiment shows that it is necessary to search for cappingconditions, under which the undesired formation of the acetylatederroneous sequence of the Nth amino acid (the last one coupled) isprevented, without the capping ability of the mixture used being reducedto such a significant extent that a potential (N−1) impurity is nolonger capped.

4.3 Variation in the Capping Conditions

The couplings of Fmoc-Arg(20)-OH, Fmoc-Leu(10)-OH, Fmoc-Gly(4)-OH andFmoc-Thr(5)-OH were investigated. Various capping conditions werecompared to one another.

The capping conditions were varied in a laboratory synthesis ofLixisenatide. Particular attention was paid to the contents of undesiredAc(N-44) and desired Ac([N−1]-44). The conditions tested are as follows:

-   -   10% acetic anhydride/5% DIPEA in DMF for 20 minutes    -   10% acetic anhydride/5% DIPEA in DMF for 10 minutes    -   2% acetic anhydride/1% DIPEA in DMF for 20 minutes    -   2% acetic anhydride/1% DIPEA in DMF for 10 minutes

The investigations were carried out at the positions Arg(20), Leu(10),Thr(5) and Gly(4). The results are compiled in tables 3-6.

The data were also compared with the result of a GMP synthesis ofLixisenatide (“GMP capping” in tables 3-6). The capping conditionscorresponded to the conditions 10% acetic anhydride/5% DIPEA in DMF. Thecontact time of the resin with the capping mixture in the GMP batch was7-8 minutes longer, and was therefore 27-28 minutes. This arose from thelonger time taken to pump the capping mixture away.

4.3.1 Coupling at Position Arg(20)

Fmoc-Arg(Pbf)-OH was coupled to Leu(21). On those chains on which nocoupling took place (product H(21-44)), the product Ac(21-44) was formedby the subsequent capping. Both products Ac(20-44) and H(20-44) areformed when, during capping, the Fmoc group is undesirably cleaved(formation of H(20-44)) and acetylation occurs (formation of Ac(2044)).

It can be clearly seen in table 3 that the degree of formation of theundesired products H(20-44) and Ac(20-44) is dependent both on thecapping time and on the amount of acetic anhydride and DIPEA (seeAc(20-44)% column). The highest percentage value can be seen in the GMPcapping. The lowest content of Ac(20-44) is found under the conditions“2% acetic anhydride/1% DIPEA in DMF for 10 minutes”.

The capping power of the various capping mixtures (and hence theoriginal intended use) is approximately the same (see column Ac(21-44)),i.e. all capping mixtures convert H(21-44)). The mixture “2% aceticanhydride/1% DIPEA in DMF for 10 minutes” also fulfils the desiredpurpose of avoiding (N−1) impurities.

TABLE 3 Results of the coupling of Fmoc-Arg(Pbf)-OH at position 20. Thetable shows the content of acetylated and non-acetylated fragmentsdepending on the capping conditions. The results were obtained by meansof LC-MS. The data were compared with the results from a GMP synthesis(“GMP capping”). Capping conditions Ac(20-44) % Fmoc(20-44) % H(20-44) %H(21-44) % Ac(21-44) % 10 min/2% acetic 0.75 96.48 0.08 0.66 2.03anhydride, 1% DIPEA 10 min/10% acetic 0.92 95.87 0.55 0.69 1.96anhydride, 5% DIPEA 20 min/2% acetic 1.63 95.83 0.14 0.55 1.85anhydride, 1% DIPEA 20 min/10% acetic 2.26 95.32 0.06 0.60 1.77anhydride, 5% DIPEA GMP capping 2.64 94.47 0.03 0.68 2.18

4.3.2 Coupling at the Positions Leu(10), Gly(4) and Thr(5)

The results for Leu(10) are given in table 4 and confirm the resultswhich were obtained for position Arg(20). The content of undesiredproducts Ac(10-44) and H(10-44), which are formed during the capping ofthe free amino groups of the product H(11-44), is lowest under theconditions “2% acetic anhydride, 1% DIPEA for 10 minutes”. The cappingpower is comparable in the different capping mixtures.

TABLE 4 Results of the coupling of Fmoc-Leu-OH at position 10. The tableshows the content of acetylated and non-acetylated fragments dependingon the capping conditions. The results were obtained by means of LC-MS.Capping conditions Ac(10-44) % Fmoc(10-44) % H(10-44) % H(11-44) %Ac(11-44) % 10 min/2% acetic 0.06 98.90 0.42 0.18 0.43 anhydride, 1%DIPEA 10 min/10% acetic 0.20 98.57 0.61 0.16 0.46 anhydride, 5% DIPEA 20min/2% acetic 0.13 98.24 0.90 0.18 0.56 anhydride, 1% DIPEA 20 min/10%acetic 0.45 98.44 0.52 0.15 0.44 anhydride, 5% DIPEA

For the coupling of Gly(4) as well, the contents of the undesiredproducts Ac(4-44) are dependent on the capping mixture and the reactiontime. The capping power is the same in the different mixtures (table 5).

TABLE 5 Results of the coupling of Fmoc-Gly-OH at position 4. The tableshows the content of acetylated and non-acetylated fragments dependingon the capping conditions. The results were obtained by means of LC-MS.Capping conditions Ac(4-44) % Fmoc(4-44) % H(4-44) % H(6-44) % Ac(5-44)% 10 min/2% acetic 0.09 98.21 0.55 0.56 0.61 anhydride, 1% DIPEA 10min/10% acetic 0.26 98.42 0.39 0.41 0.52 anhydride, 5% DIPEA 20 min/2%acetic 0.10 98.40 0.47 0.36 0.67 anhydride, 1% DIPEA 20 min/10% acetic0.39 98.02 0.43 0.39 0.77 anhydride, 5% DIPEA GMP capping 0.92 97.540.51 0.40 0.63

In addition to the positions Arg(20), Leu(10) and Gly(4), the positionThr(5) was also investigated. In contrast to the three former positions,the contents of the undesired product Ac(N-44) (Ac(5-44) at position 5)are approximately the same under the various capping conditions.However, the capping power of the different mixtures is also comparablehere (table 6).

TABLE 6 Results of the coupling of Fmoc-Thr(tBu)-OH at position 5. Thetable shows the content of acetylated and non-acetylated fragmentsdepending on the capping conditions. The results were obtained by meansof LC-MS. Capping conditions Ac(5-44) % Fmoc(5-44) % H(5-44) % H(6-44) %Ac(6-44) % 10 min/2% acetic 0.04 97.80 0.33 0.24 1.58 anhydride, 1%DIPEA 10 min/10% acetic 0.07 97.93 0.15 0.24 1.61 anhydride, 5% DIPEA 20min/2% acetic 0.03 97.69 0.36 0.23 1.70 anhydride, 1% DIPEA 20 min/10%acetic 0.03 97.70 0.42 0.29 1.55 anhydride, 5% DIPEA GMP capping 0.0797.77 0.25 0.24 1.67

4.3.3 Summary

At the positions Arg(20), Leu(10) and Gly(4), the mild capping mixture(2% acetic anhydride/1% DIPEA in DMF for 10 minutes) is sufficient inorder to maintain the desired effect of avoiding (N−1) impurities byacylation. However, in these three cases, the respective formation ofAc(20-44), Ac(10-44) and Ac(4-44) is dependent on the capping time andalso on the capping mixture. This does not apply to the position Thr(5).

EXAMPLE 5 Synthesis of Lixisenatide

The example relates to the synthesis of Lixisenatide (cf. SEQ ID NO:1).At the start of the synthesis, the solid-phase-bonded linker bears anFmoc protecting group. The individual amino acid units were coupledstarting from the C-terminus (position 44) towards the N-terminus incoupling cycles, which consist of the steps of

-   -   Fmoc cleavage    -   Coupling of the Fmoc-protected amino acid unit and    -   Capping.

At the positions Arg(20), Glu(17), Gln(13), Leu(10) and Gly(4), thecapping method according to the invention (2% acetic anhydride/1% DIPEAin DMF for 10 minutes) was used. For these positions, the instructionsfor a coupling cycle are described below. At the other positions,capping was carried out with 10% acetic anhydride/5% DIPEA in DMF for 20minutes. This capping is described, by way of example, at the positionThr(5). The capping method according to the invention comprises milderconditions.

The batch size was 1050 mmol of Rink resin.

5.1. Coupling of Fmoc-Arg(Pbf)-OH at Position 20 5.1.1 Fmoc Cleavage

7 l of DMF were added to the reactor, followed by a mixture of 7.9 l ofpiperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes,then filtered with suction. This process was repeated and stirring wascarried out for 30 minutes; then filtering with suction was carried outagain. After the Fmoc cleavage, the resin was washed 7 times in thefollowing sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l),DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor herewas filled each time with the respective washing solvent, then stirringwas carried out for 3 minutes and filtering with suction was carried outagain.

5.1.2 Coupling of Fmoc-Arg(Pbf)-OH

21 l of DMF were added to the reactor. Thereafter, 2.125 kg ofFmocArg(Pbf)-OH were weighed in and 5.3 l of DMF were added. Aftercomplete dissolution, this solution was emptied into the reactor,followed by a solution of 502 g hydroxybenzotriazole hydrate (HOBthydrate) in 2.2 l of DMF. Finally, 413 g of N,N-diisopropylcarbodiimide(DIC) were added to the reactor. The coupling time was 6-18 h. Aftercoupling, the solvent was filtered off from the resin by suction and thecapping was immediately continued.

5.1.3 Capping (According to the Invention)

The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l ofDMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine(DIPEA) were mixed in a 2 l Schott bottle and added to the resin in thereactor. The reactor was stirred for 10 minutes, then filtering withsuction was carried out. After the capping, the resin was washed 5 timesin the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l),DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time withthe respective washing solvent, then stirring was carried out for 3minutes and filtering with suction was carried out again.

5.2. Coupling of Fmoc-Glu(OtBu)-OH Hydrate at Position 17 5.2.1 FmocCleavage

7 l of DMF were added to the reactor, followed by a mixture of 7.9 l ofpiperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes,then filtered with suction. This process was repeated and stirring wascarried out for 30 min; then filtering with suction was carried outagain. After the Fmoc cleavage, the resin was washed 7 times in thefollowing sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l),DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor herewas filled each time with the respective washing solvent, then stirringwas carried out for 3 minutes and filtering with suction was carried outagain.

5.2.2 Coupling of Fmoc-Glu(OtBu)-OH Hydrate

21 l of DMF were added to the reactor. Thereafter, 1.453 kg ofFmocGlu(OtBu)-OH hydrate were weighed in and 5.3 l of DMF were added.After complete dissolution, this solution was emptied into the reactor,followed by a solution of 502 g hydroxybenzotriazole hydrate (HOBthydrate) in 2.2 l of DMF. Finally, 413 g of N,N-diisopropylcarbodiimide(DIC) were added to the reactor. The coupling time was 6-18 h. Aftercoupling, the solvent was filtered off from the resin by suction and thecapping was immediately continued.

5.2.3 Capping (According to the Invention)

The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l ofDMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine(DIPEA) were mixed in a 2 l Schott bottle and added to the resin in thereactor. The reactor was stirred for 10 minutes, then filtering withsuction was carried out. After the capping, the resin was washed 5 timesin the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l),DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time withthe respective washing solvent, then stirring was carried out for 3minutes and filtering with suction was carried out again.

5.3 Coupling of Fmoc-Gln(Trt)-OH at Position 13 5.3.1 Fmoc Cleavage

7 l of DMF were added to the reactor, followed by a mixture of 7.9 l ofpiperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes,then filtered with suction. This process was repeated and stirring wascarried out for 35 minutes; then filtering with suction was carried outagain. After the Fmoc cleavage, the resin was washed 7 times in thefollowing sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l),DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor herewas filled each time with the respective washing solvent, then stirringwas carried out for 3 minutes and filtering with suction was carried outagain.

5.3.2 Coupling of Fmoc-Gln(Trt)-OH

21 l of DMF were added to the reactor. Thereafter, 2.001 kg ofFmocGln(Trt)-OH were weighed in and 5.3 l of DMF were added. Aftercomplete dissolution, this solution was emptied into the reactor,followed by a solution of 502 g of hydroxybenzotriazole hydrate (HOBthydrate) in 2.2 l of DMF. Finally, 413 g of N,N-diisopropylcarbodiimide(DIC) were added to the reactor. The coupling time was 6-18 h. Aftercoupling, the solvent was filtered off from the resin by suction and thecapping was immediately continued.

5.3.3 Capping (According to the Invention)

The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l ofDMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine(DIPEA) were mixed in a 2 l Schott bottle and added to the resin in thereactor. The reactor was stirred for 10 minutes, then filtering withsuction was carried out. After the capping, the resin was washed 5 timesin the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l),DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time withthe respective washing solvent, then stirring was carried out for 3minutes and filtering with suction was carried out again.

5.4 Coupling of Fmoc-Leu-OH at Position 10 5.4.1 Fmoc Cleavage

7 l of DMF were added to the reactor, followed by a mixture of 7.9 l ofpiperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes,then filtered with suction. This process was repeated and stirring wascarried out for 35 minutes; then filtering with suction was carried outagain. After the Fmoc cleavage, the resin was washed 7 times in thefollowing sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l),DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor herewas filled each time with the respective washing solvent, then stirringwas carried out for 3 minutes and filtering with suction was carried outagain.

5.4.2 Coupling of Fmoc-Leu-OH

21 l of DMF were added to the reactor. Thereafter, 1.158 kg ofFmoc-Leu-OH were weighed in and 5.3 l of DMF were added. After completedissolution, this solution was emptied into the reactor, followed by asolution of 502 g hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2 lof DMF. Finally, 413 g of N,N-diisopropylcarbodiimide (DIC) were addedto the reactor. The coupling time was 6-18 h. After coupling, thesolvent was filtered off from the resin by suction and the capping wasimmediately continued.

5.4.3 Capping (According to the Invention)

The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l ofDMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine(DIPEA) were mixed in a 2 l Schott bottle and added to the resin in thereactor. The reactor was stirred for 10 minutes, then filtering withsuction was carried out. After the capping, the resin was washed 5 timesin the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l),DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time withthe respective washing solvent, then stirring was carried out for 3minutes and filtering with suction was carried out again.

5.5 Coupling of Fmoc-Gly-OH at Position 4 5.5.1 Fmoc Cleavage

7 l of DMF were added to the reactor, followed by a mixture of 7.9 l ofpiperidine in 16.6 l of DMF. This mixture was stirred for 5 minutes,then filtered with suction. This process was repeated and stirring wascarried out for 35 minutes; then filtering with suction was carried outagain. After the Fmoc cleavage, the resin was washed 7 times in thefollowing sequence: DMF (31.1 l), DMF (31.1 l), isopropanol (31.1 l),DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF (31.1 l). The reactor herewas filled each time with the respective washing solvent, then stirringwas carried out for 3 minutes and filtering with suction was carried outagain.

5.5.2 Coupling of Fmoc-Gly-OH

21 l of DMF were added to the reactor. Thereafter, 1.217 kg ofFmoc-Gly-OH were weighed in and 5.3 l of DMF were added. After completedissolution, this solution was emptied into the reactor, followed by asolution of 627 g of hydroxybenzotriazole hydrate (HOBt hydrate) in 2.2l of DMF. Finally, 517 g of N,N-diisopropylcarbodiimide (DIC) were addedto the reactor. The coupling time was 6-18 h. After coupling, thesolvent was filtered off from the resin by suction and the capping wasimmediately continued.

5.5.3 Capping (According to the Invention)

The reactor was filled with 26.3 l of DMF. At the same time, 1.2 l ofDMF, 0.53 l of acetic anhydride and 0.26 l of diisopropylethylamine(DIPEA) were mixed in a 2 l Schott bottle and added to the resin in thereactor. The reactor was stirred for 10 minutes, then filtering withsuction was carried out. After the capping, the resin was washed 5 timesin the following sequence: DMF (24 l), isopropanol (31.1 l), DMF (8 l),DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time withthe respective washing solvent, then stirring was carried out for 3minutes and filtering with suction was carried out again.

5.6 Coupling of Fmoc-Thr(tBu)-OH at Position 5

5.6.1 Fmoc Cleavage 7 l of DMF were added to the reactor, followed by amixture of 7.9 l of piperidine in 16.6 l of DMF. This mixture wasstirred for 5 minutes, then filtered with suction. This process wasrepeated and stirring was carried out for 35 minutes; then filteringwith suction was carried out again. After the Fmoc cleavage, the resinwas washed 7 times in the following sequence: DMF (31.1 l), DMF (31.1l), isopropanol (31.1 l), DMF (31.1 l), DMF (8 l), DMF (31.1 l), DMF(31.1 l). The reactor here was filled each time with the respectivewashing solvent, then stirring was carried out for 3 minutes andfiltering with suction was carried out again.

5.6.2 Coupling of Fmoc-Thr(tBu)-OH

21 l of DMF were added to the reactor. Thereafter, 1.628 kg ofFmocThr(tBu)-OH were weighed in and 5.3 l of DMF were added. Aftercomplete dissolution, this solution was emptied into the reactor,followed by a solution of 627 g of hydroxybenzotriazole hydrate (HOBthydrate) in 2.2 l of DMF. Finally, 517 g of N,N-diisopropylcarbodiimide(DIC) were added to the reactor. The coupling time was 6-18 h. Aftercoupling, the solvent was filtered off from the resin by suction and thecapping was immediately continued.

5.6.3 Capping

The reactor was filled with 10.5 l of DMF. At the same time, 15.8 l ofDMF, 3.2 l of acetic anhydride and 1.6 l of diisopropylethylamine(DIPEA) were mixed in a mixing vessel and added to the resin in thereactor. The reactor was stirred for 20 minutes, then filtering withsuction was carried out. After the capping, the resin was washed 5 timesin the following sequence: DMF (24 l), isopropanol (31.1 l), DMT (8 l),DMF (31.5 l), DMF (31.5 l). The reactor here was filled each time withthe respective washing solvent, then stirring was carried out for 3minutes and filtering with suction was carried out again.

5.7 Results

The HPLC chromatogram of the crude product of the Lixisenatide synthesiswith the capping method according to the invention at the positionsArg(20), Glu(17), Gln(13), Leu(10) and Gly(4), and capping in the othercouplings as described under 5.6.3, is shown in FIGS. 6A-6C. The peakswith the impurities acetyl(20-44), acetyl(17-44), acetyl(13-44),acetyl(10-44) and acetyl(4-44)/acetyl(6-44) are indicated.

5.8 Comparison

The capping steps of all couplings, as described under 5.6.3, werecarried out, leading to increased formation of the undesired erroneoussequences Ac(20-44), Ac(17-44), Ac(13-44), Ac(10-44) andAc(4-44)/Ac(6-44). The HPLC chromatogram of a crude Lixisenatide fromthis test is shown in FIG. 6A.

FIG. 6B shows a HPLC chromatogram of crude Lixisenatide, synthesizedwith the capping method according to the invention at the positionsArg(20), Glu(17), Gln(13), Leu(10) and Gly(4).

FIG. 6C shows the superimposition of the HPLC chromatograms from FIGS.6A and B. It is apparent that the synthesis of Lixisenatide using thecapping method according to the invention in batch operation led to adistinct reduction in the erroneous sequences Ac(20-44), Ac(17-44),Ac(13-44), Ac(10-44) and Ac(4-44)/Ac(6-44).

By using a milder capping mixture (2% acetic anhydride/1% DIPEA in DMFfor 10 minutes), it was possible to reduce the level of acetylatederroneous sequences of Ac(20-44), Ac(17-44), Ac(13-44), Ac(10-44) andAc(4-44) in the crude product of Lixisenatide or eliminate themtherefrom. Since a Lixisenatide crude product which was prepared by thecapping according to the invention included the acetylated by-productsAc(17-44), Ac(13-44) and Ac(10-44) in particular in considerably reducedamounts, the purification of Lixisenatide was simplified. As a result,pooling of the fractions after the first preparative chromatography runof Lixisenatide gave more fractions which met the specification criteriaand thus did not have to be discarded. This led to an improved yield.

EXAMPLE 6 Capping at 9 Specific Positions in the Synthesis ofLixisenatide

As discussed in Example 5, the use of “mild” capping conditions in thesynthesis of lixisenatide at positions Arg(20), Glu(17), Gln(13),Leu(10) or/and Gly(4) could improve the profile of undesiredby-products.

This Example describes the influence of capping conditions upon theformation of acetylated and non-acetylated by-products. Variations inthe temperature (15° C., room temperature [20° C.−23° C.], 30° C.),capping duration and the ingredients of the capping composition wereperformed:

-   -   no capping,    -   mild capping conditions: 10 min capping with 2% acetic anhydride        and 1% of DI PEA (diisopropylethylamine)    -   “normal” capping conditions: 20 min capping with 10% acetic        anhydride and 5% of DIPEA    -   40 min capping with 10% acetic anhydride and 5% of DIPEA

Capping conditions of the present invention are the “mild conditions”.These conditions were used in Example 5. These conditions were found tobe advantageous.

At the 9 positions selected in this Example, acetylated sequences areobtained at capping of the (N−1) position (FIG. 7). Additionally,undesired removal of the Fmoc group at the amino acid building block mayoccur during the capping step. The unprotected amino group may beacetylated by the capping reagent or capping composition. In thisrespect, improved capping conditions may avoid the undesired cleavage ofthe Fmoc group.

6.1 Capping at Position 36/35, after Coupling of the Dipeptide BuildingBlock Pro-Pro, (36-44)

Peptide Fmoc-(36-44)-AVE0010 was produced by solid phase synthesis. Theresin was dried in divided into 4 portions. Each portion underwent oneof the four capping procedures described above at room temperature (20°C.-23° C.). Samples were dried, and the peptide was cleaved from theresin. This procedure was repeated, wherein capping was performed at 15°C. or 30° C.

In a total 12 peptide samples were obtained. The 12 peptide samples wereanalyzed with LCMS. Molecular weights were determined from the TIC(total ion current). The molecular weights of the following compoundswere determined:

TABLE 7 Ac(36-44) can be formed by Fmoc cleavage during capping andsubsequent acetylation (undesired by-product) Fmoc(36-44) desiredproduct (main product) of solid phase synthesis (36-44) can be formed byFmoc cleavage during capping, but no acetylation takes place (undesiredby-product) (38-44) may be still present if coupling of theFmoc-dipeptide building block was incomplete, but no acetylation takesplace during the capping step (undesired by-product) Ac(38-44) desiredcapping product, may be formed by capping if coupling of theFmoc-dipeptide building block was incomplete.

Table 8 shows the content of products obtained after Fmoc-ProProcoupling and subsequence capping (% of total peptide content).

Ac(36-44) Fmoc(36-44) (36-44) (38-44) Ac(38-44) Position 36/35 Pro-Pro,15° C. without capping 0.02 99.96 0 0.02 0 10 min, 2% Ac2O, 1% DIPEA0.37 99.6 0 0.03 0 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.09 99.73 0 0.18 0(normal) 40 min, 10% Ac2O, 5% DIPEA 0.4 99.57 0 0.02 0 Position 36/35Pro-Pro, RT without capping 0.09 99.84 0 0 0.06 10 min, 2% Ac2O, 1%DIPEA 0.47 99.47 0 0 0.06 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.9 99.04 00 0.06 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.52 98.42 0 0 0.05 Position36/35 Pro-Pro, 30° C. without capping 0 100 0 0 0 10 min, 2% Ac2O, 1%DIPEA 0.21 99.79 0 0 0 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.66 99.34 0 00 (normal) 40 min, 10% Ac2O, 5% DIPEA 2.39 97.61 0 0 0

The results are described in FIG. 8. Compounds (36-44), (38-44) andAc(38-44) were not found, or were found in small amounts. The amount ofthe undesired product Ac(36-44) increases with the strength of thecapping cocktail and capping duration in most cases. The amount of thisproduct increases with temperature.

6.2 Capping at Position 23, after Coupling of the Building Ile, (23-44)

The synthesis of Fmoc(23-44) was performed as described in section 6.1.Experiments at 15° C./30° C. and at room temperature were performed withdifferent batches.

Table 9 shows the content of products obtained after Fmoc-Ile couplingand subsequence capping (% of total peptide content)

Ac(23-44) Fmoc(23-44) (23-44) (24-44) Ac(24-44) Position 23 Ile, 15° C.without capping 0 99.7 0 0.16 0.14 10 min, 2% Ac2O, 1% DIPEA 0 99.560.18 0.11 0.15 (mild) 20 min, 10% Ac2O, 5% DIPEA 0 99.57 0.2 0.11 0.13(normal) 40 min, 10% Ac2O, 5% DIPEA 0 99.59 0.16 0.1 0.15 Position 23Ile, RT without capping 0 99.81 0 0.19 0 10 min, 2% Ac2O, 1% DIPEA 0.1399.7 0 0.16 0 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.26 99.54 0 0.2 0(normal) 40 min, 10% Ac2O, 5% DIPEA 0.61 99.21 0 0.18 0 Position 23 Ile,30° C. without capping 0 99.66 0 0.18 0.16 10 min, 2% Ac2O, 1% DIPEA 0.199.38 0.19 0.16 0.17 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.77 98.65 0.250.15 0.17 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.43 98.15 0.16 0.12 0.14

The results are described in FIG. 9. Depending upon the capping reagentat RT and 30° C., the content of undesired compound Ac(23-44) increases.“Normal” capping at 20° C. results in 0.26% of Ac(23-44). Prolongationof capping (40 min instead of 20 min) has a negative impact on theAc(23-44) content.

Formation of the desired product Ac(24-44) is independent from thecapping composition.

6.3 Capping at Position 21, after Coupling of the Building Block Leu,(21-44)

The synthesis of Fmoc(21-44) was performed as described in section 6.1.Experiments at 15° C./30° C. and at room temperature were performed withdifferent batches.

Table 10 shows the content of products obtained after Fmoc-Leu couplingand subsequence capping (% of total peptide content)

Ac(21-44) Fmoc(21-44) (21-44) (22-44) Ac(22-44) Position 21 Leu, 15° C.without capping 0 99.91 0 0 0.09 10 min, 2% Ac2O, 1% DIPEA 0.03 99.780.07 0 0.12 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.09 99.74 0.05 0 0.12(normal) 40 min, 10% Ac2O, 5% DIPEA 0.36 99.48 0.03 0 0.13 Position 21Leu, RT without capping 0 99.77 0 0.07 0.16 10 min, 2% Ac2O, 1% DIPEA0.06 99.64 0 0.14 0.16 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.2 99.62 00.04 0.14 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.46 99.34 0 0.04 0.16Position 21 Leu, 30° C. without capping 0 99.86 0.04 0 0.11 10 min, 2%Ac2O, 1% DIPEA 0.1 99.67 0.11 0 0.12 (mild) 20 min, 10% Ac2O, 5% DIPEA0.86 98.95 0.06 0 0.13 (normal) 40 min, 10% Ac2O, 5% DIPEA 2.57 97.220.05 0 0.16

The results are described in FIG. 10. The content of undesired compoundAc(21-44) is largest at “40 min, 10% Ac2O, 5% DIPEA” at 15° C., RT and30° C. The content of compound Ac(21-44) increases with temperature.

Formation of the desired compound Ac(22-44) is independent from thecapping composition. Even without capping, this compound is formed.

6.4 Capping at Position 19, after Coupling of the Building Block Val,(19-44)

The synthesis of Fmoc(19-44) was performed as described in section 6.1.Experiments at 15° C./30° C. and at room temperature were performed withdifferent batches.

Table 11 shows the content of products obtained after Fmoc-Val couplingand subsequence capping (% of total peptide content)

Ac(19-44) Fmoc(19-44) (19-44) (20-44) Ac(20-44) Position 19 Val, 15° C.without capping 0 98.98 0.13 0.46 0.44 10 min, 2% Ac2O, 1% DIPEA 0.1198.79 0.44 0.25 0.4 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.56 98.68 0.140.25 0.37 (normal) 40 min, 10% Ac2O, 5% DIPEA 1 98.17 0.09 0.23 0.51Position 19 Val, RT without capping 0 99.61 0 0.23 0.16 10 min, 2% Ac2O,1% DIPEA 0.14 99.52 0 0.15 0.2 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.4399.23 0 0.17 0.17 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.9 98.9 0 0 0.2Position 19 Val, 30° C. without capping 0 99.16 0.08 0.4 0.36 10 min, 2%Ac2O, 1% DIPEA 0.41 98.59 0.4 0.27 0.33 (mild) 20 min, 10% Ac2O, 5%DIPEA 2.3 96.89 0.14 0.22 0.45 (normal) 40 min, 10% Ac2O, 5% DIPEA 5.0994.1 0.11 0.22 0.48

The results are described in FIG. 11. The content of compound Ac(19-44)is largest at “40 min, 10% Ac2O, 5% DIPEA” at 15° C., RT and 30° C. Thecontent of compound Ac(19-44) increases with temperature.

Formation of the desired compound Ac(20-44) increases with the strengthof the capping composition. The content of undesired (20-44) decreaseswith increasing strength of the capping composition.

6.5 Capping at Position 18, after Coupling of the Building Block Ala,(18-44)

The synthesis of Fmoc(18-44) was performed as described in section 6.1.Experiments at 15° C./30° C. and at room temperature were performed withdifferent batches.

Table 12 shows the content of products obtained after Fmoc-Ala couplingand subsequence capping (% of total peptide content)

Ac(18-44) Fmoc(18-44) (18-44) (19-44) Ac(19-44) Position 18 Ala, 15° C.without capping 0 98.77 0.48 0.33 0.42 10 min, 2% Ac2O, 1% DIPEA 0.4898.24 0.53 0.26 0.49 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.76 98.18 0.270.2 0.59 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.12 97.91 0.23 0.22 0.52Position 18 Ala, RT without capping 0 99.63 0 0 0.37 10 min, 2% Ac2O, 1%DIPEA 0.1 99.43 0.1 0 0.36 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.77 98.690.18 0 0.36 (normal) 40 min, 10% Ac2O, 5% DIPEA 0.38 99.28 0 0 0.38Position 18 Ala, 30° C. without capping 0 98.76 0.53 0.2 0.5 10 min, 2%Ac2O, 1% DIPEA 0.92 98.07 0.32 0.11 0.58 (mild) 20 min, 10% Ac2O, 5%DIPEA 2.44 96.67 0.09 0.14 0.65 (normal) 40 min, 10% Ac2O, 5% DIPEA 6.3392.73 0.05 0.14 0.73

The results are described in FIG. 12. The content of undesired compoundAc(18-44) is largest at “40 min, 10% Ac2O, 5% DIPEA” at 15° C. and 30°C. The content of compound Ac(18-44) increases with temperature increasefrom 15° C. to 30° C.

Formation of the desired compound Ac(19-44) increases at 15° C. and 30°C. with the strength of the capping composition.

6.6 Capping at Position 15, after Coupling of the Building Block Glu(15-44)

The synthesis of Fmoc(15-44) was performed as described in section 6.1.Experiments at 15° C./30° C. and at room temperature were performed withdifferent batches.

Table 13 shows the content of products obtained after Fmoc-Glu couplingand subsequence capping (% of total peptide content)

Ac(15-44) Fmoc(15-44) (15-44) (16-44) Ac(16-44) Position 15 Glu, 15° C.without 0 99.28 0 0.59 0.13 capping 10 min, 2% 0.05 99.08 0.15 0.57 0.15Ac2O, 1% DIPEA (mild) 20 min, 10% 0.19 99.08 0 0.58 0.15 Ac2O, 5% DIPEA(normal) 40 min, 10% 0.39 98.82 0 0.63 0.16 Ac2O, 5% DIPEA Position 15Glu, RT without 0 99.72 0.12 0 0.17 capping 10 min, 2% 0.1 99.4 0.36 00.16 Ac2O, 1% DIPEA (mild) 20 min, 10% 0.42 99.13 0.2 0.04 0.21 Ac2O, 5%DIPEA (normal) 40 min, 10% 0.89 98.65 0.22 0.05 0.19 Ac2O, 5% DIPEAPosition 15 Glu, 30° C. without 0 98.93 0 0.91 0.16 capping 10 min, 2%0.17 98.7 0 0.95 0.18 Ac2O, 1% DIPEA (mild) 20 min, 10% 1.62 97.3 0 0.880.2 Ac2O, 5% DIPEA (normal) 40 min, 10% 3.24 95.63 0 0.94 0.19 Ac2O, 5%DIPEA

The results are described in FIG. 13. The content of undesired compoundAc(15-44) is largest at “40 min, 10% Ac2O, 5% DIPEA” at 15° C., RT and30° C. The content of compound Ac(15-44) increases with temperature.

Formation of the desired compound Ac(16-44) is independent from thecapping composition. Even without capping, this compound is formed.

6.7 Capping at Position 12, after Coupling of the Building Block Lys(12-44)

The synthesis of Fmoc(12-44) was performed as described in section 6.1.Experiments at 15° C./30° C. and at room temperature were performed withdifferent batches.

Table 14 shows the content of products obtained after Fmoc-Lys couplingand subsequence capping (% of total peptide content)

Ac(12-44) Fmoc(12-44) (12-44) (13-44) Ac(13-44) Position 12 Lys, 15° C.without capping 0 99.43 0.13 0 0.44 10 min, 2% Ac2O, 1% DIPEA 0.15 99.250.17 0 0.43 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.3 99.03 0.17 0 0.49(normal) 40 min, 10% Ac2O, 5% DIPEA 0.55 98.88 0.16 0 0.41 Position 12Lys, RT without capping 0 99.12 0 0.17 0.71 10 min, 2% Ac2O, 1% DIPEA 099.29 0 0 0.71 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.5 98.76 0 0 0.74(normal) 40 min, 10% Ac2O, 5% DIPEA 1.12 98.15 0 0 0.73 Position 12 Lys,30° C. without capping 0 99.41 0.15 0 0.44 10 min, 2% Ac2O, 1% DIPEA0.35 99.02 0.16 0 0.47 (mild) 20 min, 10% Ac2O, 5% DIPEA 1.55 97.89 0.140 0.41 (normal) 40 min, 10% Ac2O, 5% DIPEA 3.53 95.87 0.16 0 0.44

The results are described in FIG. 14. The content of undesired compoundAc(12-44) is largest at “40 min, 10% Ac2O, 5% DIPEA” at 15° C., RT and30° C. The content of compound Ac(12-44) increases with temperature.

Formation of the desired compound Ac(13-44) is independent from thecapping composition. Even without capping, this compound is formed.

6.8 Capping at Position 8, after Coupling of the Building Block Ser(8-44)

The synthesis of Fmoc(8-44) was performed as described in section 6.1.Experiments at 15° C., RT and 30° C. were performed with the same batch.

Table 15 shows the content of products obtained after Fmoc-Ser couplingand subsequence capping (% of total peptide content)

Ac(8-44) Fmoc(8-44) (8-44) (9-44) Ac(9-44) Position 8 Ser, 15° C.without capping 0 100 0 0 0 10 min, 2% Ac2O, 1% DIPEA 0 99.79 0 0 0.21(mild) 20 min, 10% Ac2O, 5% DIPEA 0.29 99.53 0 0 0.18 (normal) 40 min,10% Ac2O, 5% DIPEA 1.08 98.72 0 0 0.21 Position 8 Ser, RT withoutcapping 0 99.67 0 0.18 0.16 10 min, 2% Ac2O, 1% DIPEA 0.22 99.78 0 0 0(mild) 20 min, 10% Ac2O, 5% DIPEA 1.12 98.88 0 0 0 (normal) 40 min, 10%Ac2O, 5% DIPEA 2.1 97.9 0 0 0 Position 8 Ser, 30° C. without capping 0100 0 0 0 10 min, 2% Ac2O, 1% DIPEA 0.29 99.27 0.27 0 0.18 (mild) 20min, 10% Ac2O, 5% DIPEA 2.1 97.8 0 0 0.1 (normal) 40 min, 10% Ac2O, 5%DIPEA 5.02 94.74 0 0 0.24

The results are described in FIG. 15. The content of undesired compoundAc(8-44) is largest at “40 min, 10% Ac2O, 5% DIPEA” at 15° C., RT and30° C. The content of compound Ac(8-44) increases with temperature.

6.9 Capping at Position 6, after Coupling of the Building Block Phe(6-44)

The synthesis of Fmoc(86-44) was performed as described in section 6.1.Experiments at 15° C., RT and 30° C. were performed with the same batch.

Table 16 shows the content of products obtained after Fmoc-Phe couplingand subsequence capping (% of total peptide content)

Ac(6-44) Fmoc(6-44) (6-44) (7-44) Ac(7-44) Position 6 Phe, 15° C.without capping 0 99.21 0 0.38 0.41 10 min, 2% Ac2O, 1% DIPEA 0 99 0.390.28 0.34 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.35 98.73 0.32 0.26 0.33(normal) 40 min, 10% Ac2O, 5% DIPEA 0.62 98.6 0.3 0.18 0.3 Position 6Phe, RT without capping 0 99.24 0 0.39 0.37 10 min, 2% Ac2O, 1% DIPEA0.2 98.68 0.6 0.25 0.28 (mild) 20 min, 10% Ac2O, 5% DIPEA 0.57 98.490.31 0.25 0.38 (normal) 40 min, 10% Ac2O, 5% DIPEA 1.32 97.9 0.33 0.20.24 Position 6 Phe, 30° C. without capping 0 99.24 0 0.43 0.33 10 min,2% Ac2O, 1% DIPEA 0.33 98.36 0.55 0.29 0.46 (mild) 20 min, 10% Ac2O, 5%DIPEA 1.54 97.42 0.37 0.3 0.37 (normal) 40 min, 10% Ac2O, 5% DIPEA 3.7395.91 0 0 0.36

The results are described in FIG. 16. The content of undesired compoundAc(6-44) is largest at “40 min, 10% Ac2O, 5% DIPEA” at 15° C., RT and30° C. The content of compound Ac(6-44) increases with temperature.

Formation of the desired compound Ac(7-44) is independent from thecapping composition. Even without capping, this compound is formed.

Temperature has only slight influence on formation of the desiredcompound Ac(7-44). The content of undesired (7-44) decreases withincreasing strength of the capping composition.

6.10 Summary

Undesired formation of Ac(X-44)-compound strongly depends upon thecapping duration, the capping composition and the capping temperature.With increasing capping duration, increasing capping temperature, andincreased content of acetic anhydride and DIPEA in the cappingcomposition the content of undesired Ac(X-44) compound increases.

6.11 Capping Under “Normal” Conditions, Depending Upon Temperature.

FIGS. 17 and 18 summarize the data obtained in capping at differenttemperatures at the 9 positions in the synthesis of Lixisenatide under“normal” conditions “20 min, 10% Ac2O, 5% DIPEA”, as described in thisExample.

FIG. 17 shows a comparison of GMP capping of Ac(X-44), depending onreaction temperature. Values given for 15° C. and 30° C. are positiveand negative deviations from “room temperature” values (grey area).

The formation of undesired product Ac(X-44) is ≥0.5% in 5 of 9positions, in 3 positions between 0.5% and 1%, and in only oneposition >1%. A large increase is observed at 30° C., while at 15° C.,formation of Ac(X-44) slightly decreases.

This means that GMP capping “20 min, 10% Ac2O, 5% DIPEA” can beperformed at different positions between 15° C. and room temperature,which can be 20-23° C.

FIG. 18 shows a comparison of GMP capping of Ac[(X−1)-44], depending onreaction temperature. Values given for 15° C. and 30° C. are positiveand negative deviations from “room temperature” values (grey area)

Regarding the desired formation of the Ac[(X−1)-44] compounds at RT, thedeviation at 15° C. is between +0.23 und -0.25%. At 30° C., thedeviation is between +0.29 und −0.33%. Formation of the desired cappingproduct Ac[(X−1)-44] is thus less dependent upon the temperature thanthe undesired formation of Ac(X-44).

At 15° C. and 30° C., negative deviations of the content of desiredcompound Ac[(X−1)-44] are observed in view of capping at roomtemperature. This means that capping with “normal” conditions should beperformed at room temperature.

6.12 Capping with Different Capping Compositions at Room Temperature.

FIGS. 19 and 20 summarize the data obtained in capping with differentcapping compositions at room temperature at the 9 positions in thesynthesis of lixisenatide, as described in this Example.

FIG. 19 shows a comparison of Ac(X-44) content, depending upon thecapping composition at room temperature. Values given for “no capping”,“mild” and “40 min” conditions are positive and negative deviations from“normal capping” values (grey area).

Formation of undesired product Ac(X-44) under “20 min, 10% Ac2O, 5%DIPEA” and “40 min, 10% Ac2O, 5% DIPEA” is largest. Formation ofAc(X-44) under “normal” conditions (40 min, 10% Ac2O, 5% DIPEA) isbetween 0.2% and 1.12%. A strong decrease is observed at mild cappingconditions.

FIG. 20 shows a comparison of Ac[(X−1)-44] content, depending upon thecapping composition at room temperature. Values given for “no capping”,“mild” and “40 min” are positive and negative deviations from “normalcapping” values (grey area).

The formation of the desired product Ac[(X−1)-44] at the conditions “nocapping”, “mild” and “40 min” is within −0.14% and +0.16% in view of the“normal” conditions.

In particular, under “mild” conditions (10 min, 2% Ac2O, 1% DIPEA) ofthe invention, sufficient capping can be achieved in the synthesis oflixisenatide.

In summary, mild capping conditions, in particular capping for 10 minwith 2% Ac2O and 1% DIPEA in a solvent, are advantageous in the solidphase synthesis of lixisenatide, as described herein.

If capping is omitted after coupling at certain amino acid positions,undesired by-products comprising an incomplete amino acid sequence andbeing present in small amount, may be difficult to remove during thepurification process.

EXAMPLE 7

Cleavage of Lixisenatide from the Solid Phase

This example relates to the cleavage according to the invention ofLixisenatide from a solid phase. A solid phase (Rink resin) wasprovided, to which the peptide Lixisenatide was bonded. The peptide wassynthesized on the resin by stepwise coupling of amino acid units.

As comparative test, a cleavage according to the prior art (King et al.,Int. J. Peptide Protein Res. 1990, 36: 255-266) was carried out.

The cleavage method according to the invention is distinguished from themethod of the prior art by the following changes:

-   -   Reaction temperature from 20° C. to 26° C.    -   Number of components in the cleavage mixture reduced from 5 to 2        constituents, combined with increase in the ratio of resin to        cleavage mixture used.

TABLE 17 Comparison of the cleavage method according to the inventionand the cleavage according to the prior art. The differences areindicated in bold/underlined. Comparative process (prior art) Methodaccording to the invention Cleavage mixture [g or ml/g “peptide onresin”]: Cleavage mixture [ml/g “peptide on resin”]: a) 0.5 g phenol a)  0.25   ml   1,2-ethanedithiol b)   0.5   ml   thioanisole b)   8.25  ml   TFA c)   0.25   ml   1,2-ethanedithiol d)   0.5   ml   water e)  8.25   ml   TFA (“King's cocktail”) Cleavage mixture is cooled to 5-10°C. and Cleavage mixture is cooled to 5-10° C. and added toLixisenatide-resin(1-44) added to Lixisenatide-resin(1-44) Reactionmixture heated to 20°   C. and Reaction mixture heated to 26°   C. andstirred for 4 h stirred for 4 h Reaction mixture filtered Reactionmixture filtered Subsequent cleavage Subsequent cleavage The resinfiltered off is added to TFA The resin filtered off is added to TFA (10ml per g of resin), stirred for 1 h and (10 ml per g of resin), stirredfor 1 h and the resin is filtered off the resin is filtered off Filtratepurified and solution concentrated Filtrate purified and solutionconcentrated by distillation under reduced pressure at by distillationunder reduced pressure at 35-40° C. to at least 1/16th of the original35-40° C. to at least 1/16th of the original volume. volume. CrudeLixisenatide precipitated by addition Crude Lixisenatide precipitated byaddition of the concentrate to 6 times the volume of of the concentrateto 6 times the volume of DIPE DIPE The precipitate is resuspended twicein The precipitate is resuspended twice in ethyl acetate and filteredoff. ethyl acetate and filtered off. The precipitate is dried and thecrude The precipitate is dried and the crude Lixisenatide is isolated.Lixisenatide is isolated.

By using the cleavage according to the invention, compared to thecleavage according to the prior art, it was possible to increase theyields of the crude Lixisenatide by approximately 5% (from 20% to 25%),while the impurities profile was only slightly altered.

The method of this example is suitable for scale-up to the pilot-plantand production scale.

Table 18 summarizes the results obtained in the comparative process (seeTable 17). Three different batches 2E002, 2B008 and 2B006 oflixisenatide-resin (1-44) were used. Means and standard deviation arecalculated for each batch separately. Comparison between differentcleavage conditions should be made in tests using the same batch oflixisenatide-resin (1-44). In different batches, the solid phasesynthesis may have an impact on the yield. If not otherwise indicated,10 g of lixisenatide-resin(1-44) were used as starting material.

TABLE 18 Content of lixisenatide in the resin, and yield of lixisenatideafter cleavage of lixisenatide from the resin under standard conditions(comparative process, see Table 17). Batch of Output Number oflixisenatide- weight Content Yield experiment resin(1-44) [g] [%] [%]Batch 2E002 71002-002 2E002 2.60 22.9 10.2 71002-003 2E002 3.36 23.113.4 71002-012 2E002 1.88 20.3 6.6 separate subsequent 0.77 25.0 3.3cleavage 70609-068 2E002 3.01 21.5 11.1 71002-035 2E002 3.08 16.3 8.671002-036 2E002 3.07 16.3 8.6 70586-043 2E002 2.40 24.3 10.1 71003-0032E002 2.50 24.0 10.3 Mean ± standard 10.3 ± 1.5 deviation Batch 2B00870586-052 2B008 2.84 26.9 14.7 71001-006 2B008 3.03 20.0 11.7 71002-0482B008 2.89 22.8 12.7 71001-016 2B008 3.41 21.6 14.2 Mean ± standard 13.4± 1.4 deviation Batch 2B006 70586-056 2B006 3.02 25.0 14.3

7.1 Cleavage Yield Depending Upon the Cleavage Temperature Between 20°C. and 35° C.

Cleavage from the lixisenatide-resin(1-44) was performed under standardconditions (comparative process, see Table 17) for 4 h.

TABLE 19 Cleavage yield depending upon temperature Batch of Number oflixisenatide- Temperature Duration Yield experiment resin(1-44) [° C.][h] [%] Standard 2E002 20 4 10.3 ± 1.5 70586-050 2E002 23 4 11.770586-044 2E002 26 4 14.8 70586-046 2E002 30 4 14.2 70586-049 2E002 35 412.4

Results: The yield of lixisenatide after cleavage under standardconditions increases with increasing temperature until the optimum ofabout 26° C. Surprisingly, an increase of temperature from 23° C. to 26°C. results in a significant increase in yield.

7.2 Cleavage Yield Depending Upon the Cleavage Duration

Cleavage from the lixisenatide-resin(1-44) was performed under standardconditions (comparative process, see Table 17) at 20° C.

TABLE 20 Cleavage yield depending upon the cleavage duration Batch ofNumber of lixisenatide- Temperature Duration Yield experimentresin(1-44) [° C.] [h] [%] Standard 2E002 20 4 10.3 ± 1.5 71002-0372E002 20 6 11.3 71002-038 2E002 20 8 13.4 70586-037, 2E002 20 12 13.1 ±0.9 71003-002, 71003-004

Results: The yield of lixisenatide increases with increased cleavageduration. A maximum yield is reached after about 8 h cleavage.

7.3 Cleavage Yield Depending Upon the Temperature at Cleavage Durationof 12 h

Cleavage from the lixisenatide-resin(1-44) was performed under standardconditions (comparative process, see Table 17) for 4 h.

TABLE 21 Cleavage yield depending upon the temperature at cleavageduration of 12 h Batch of Number of lixisenatide- Temperature DurationYield experiment resin(1-44) [° C.] [h] [%] 70586-040 2E002 17 12 10.770586-037 2E002 20 12 14.1 70586-039 2E002 23 12 13.0 70586-045 2E002 2612 14.0 70586-047 2E002 30 12 12.1

Results: The yield increases at a cleavage duration of 12 h if reactiontemperature is increased. A maximum yield is obtained at 26° C., asdescribed in Example 7.1 for 4 h cleavage. Tests 70586-044 (4 h, 26° C.,Example 7) and 70586-045 (12 h, 26° C.) resulted in similar yields(14.8% vs. 14.0%).

7.4 Cleavage Yield Depending Upon the Cleavage Temperature Up to 20° C.

Cleavage from the lixisenatide-resin(1-44) was performed under standardconditions (comparative process, see Table 17) for 4 h.

TABLE 22 Cleavage yield depending upon the cleavage temperature up to20° C. Batch of Number of lixisenatide- Temperature Duration Yieldexperiment resin(1-44) [° C.] [h] [%] 71002-028 2E002 0-5° C. 21.5 6.071002-029 2E002 8-13° C. 28 8.7 71002-030 2E002 8-13° C. 40.8 11.270586-040 2E002 17° C. 12 10.7 Standard 2E002 20° C. 4 10.3 ± 1.570586-037, 2E002 20° C. 12 13.1 ± 0.9 71003-002, 71003-004

Results: The cleavage at a temperature below 20° C. requires longercleavage durations, as expected, to reach the yield obtained by cleavageat 20° C. for 4 h (standard conditions, comparative process, Table 17).

7.5 Modified Cleavage Cocktail

The standard process uses a cleavage cocktail containing fivecomponents: phenol, thioanisole, 1,2-ethandithiole, water and TFA.Subject of the example are simplified cleavage cocktails, omitting oneto three of thioanisole, phenol and water. The yield of lixisenatidecleavage from lixisenatide-resin(1-44) is determined. The “nomodification” cocktail is described in Table 17, “Comparative process”.

TABLE 23 Modified cleavage cocktail Batch of Modification of Number oflixisenatide- cleavage Yield experiment resin(1-44) composition [%]Standard 2E002 no modification 10.3 ± 1.5 71002-010 2E002 withoutthioanisole 10.7 71002-009 2E002 without phenol 12.1 71002-006 2E002without water 13.2 71002-008 2E002 without phenol and 13.3 water71003-008 2E002 water content is 13.3 reduced to 2.5% w/w 71002-0422E002 without thioanisole, 12.7 phenol and water, i.e. only TFA and1,2-ethanedithiol

Results: Omission of one or more components results in an increasedyield, except test 71002-010 (omission of thioanisole).

A simplified cleavage mixture (cleavage cocktail) has severaladvantages:

(a) simplification of analytics and quality control,(b) reduced costs,(c) facilitated handling in the production process.

7.6 TFA and 1,2-Ethanedithiol Content in the Cleavage Cocktail

Starting from test 71002-042, the influence of the TFA:1,2-ethanedithiolratio upon cleavage yield was investigated:

TABLE 24 Different TFA and 1,2-ethanedithiol ratio in the cleavagecocktail Volume in mL of Batch of TFA and 1,2- Number of Lixisenatide-ethanedithiol per g Yield Experiment resin (1-44) _(“)peptide on resin”[%] Standard 2E002 10.3 ± 1.5 71002-045 2E002 8:2  6.5 71002-043 2E0029:1  9.3 71002-044 2E002  9:0.5 11.0 71002-042 2E002 8.25:0.25 12.7Standard 2B008 13.4 ± 1.4 71002-046 2B008 8.25:0.25 15.6 71002-047 2B0088.25:0.25 14.3

Results: An increase in the 1,2-ethanedithiol content results in asignificant decrease of lixisenatide yield. The TFA:1,2-ethanedithiolratio of 8.25:0.25 was found to be the ratio with largest yield (batch2E002). This finding was confirmed by to experiments using batch 213008.

7.7 Volume of the Cleavage Cocktail

The influence of volume (and thus concentration) of the cleavagecocktail was investigated

TABLE 25 Cleavage yield, depending upon volume of the cleavage cocktail.Batch of Reduction Number of Lixisenatide- of volume Yield Experimentresin (1-44) [%] [%] Standard 2E002  0% 10.3 ± 1.5 71002-026 2E002 −10%13.3 71002-040 2E002 −15% 10.3 71002-025 2E002 −25% 11.0 70609-069 2E002−30% 10.5 71002-031 2E002 −50% 7.9

Results: The reduction of up to 30% has no influence upon cleavageyield. Larger volume reductions lead to a decreased yield.

7.8 Swelling of the “Peptide on Resin” with a Co-Solvent (Toluol orCH₂Cl₂) Before Cleavage

The rationale behind this experiment is the finding that cleavage oflixisenatide from the resin may result in an increase in temperature ofup to 5-8° C., which may lead to formation of undesired by-products andpotentially has a negative impact upon stability and thus the cleavageyield. Swelling of the “peptide on resin” in an organic solvent mayreduce the exotherm and thus may increase the yield.

TABLE 26 Cleavage yield, depending upon the presence of a co-solvent.Batch of Swelling Increase of Number of Lixisenatide- with organicDuration temperature Yield Experiment resin (1-44) solvent [h] [° C.][%] Standard 2E002 without 5-8° C. 10.3 ± 1.5 71002-016 2E002 30 mltoluol* 4 h 1-2° C. 9.8 71002-019 2E002 30 ml toluol 6 h 1-2° C. 6.171002-021 2E002 30 ml toluol 17 h 1-2° C. 9.3 71002-017 2E002 50 mltoluol 28 h 1-2° C. 4.7 71002-024 2E002 30 ml CH₂Cl₂ 24 h 1-2° C. 7.3*The total volume of TFA and toluol/CH₂Cl₂ is kept constant.

Results: Swelling with an organic co-solvent does not increase thecleavage yield.

7.9 Concentration in the Presence of a Co-Solvent

The presence of a co-solvent, having a higher boiling point than TFA,and in which lixisenatide is insoluble, may increase the yield aftercleavage from the resin, because during distillation of TFA from thefiltrate, the presence of the co-solvent may lead to precipitation oflixisenatide, and therefore can prevent the degradation of lixisenatideduring cleavage in King's cocktail.

TABLE 27 Cleavage yield, depending upon the presence of a co-solventduring TFA distillation. Batch of Number of lixisenatide- YieldExperiment resin(1-44) Solvent [%] Standard 2E002 Ohne 10.3 ± 1.571002-004 2E002 Toluol 12.0 71002-014 2E002 n-Heptan 11.1

Results: The presence of toluol in the distillation of the filtrateafter cleavage of lixisenatide from the resin leads to a slightlyincreased yield.

7.10 Optimized Cleavage Procedure of the Invention

Based upon the above-described results obtained in this Example,optimized cleavage conditions as follows were selected and tested:

-   (a) reaction temperature of 26° C.,-   (b) cleavage cocktail consists of TFA and 1,2-ethanedithiol. The    cocktail contained about 97% of TFA and about 3% of    1,2-ethanedithiol. An amount of 8.25 ml/g “peptide on resin” of TFA    and 0.25 ml/g “peptide on resin” of 1,2-ethanedithiol was used.

The cleavage yield of this cocktail, compared with the standardcomparative cocktail, was tested in batches 2E002 and 2B008.

TABLE 28 Optimized cleavage procedure of the invention Batch ofModification Number of Lixisenatide- of cleavage Yield experiment resin(1-44) composition [%] Standard 2E002 no 10.3 ± 1.5 modification70586-051 2E002 26° C., only 14.9 TFA and EDT 71001-012 2E002 26° C.,only 15.8 TFA and EDT Mean ± standard 15.4 ± 0.6 deviation Standard2B008 no 13.4 ± 1.4 modification 70586-051 2B008 26° C., only 18.5 TFAand EDT 71001-013 2B008 26° C., only 19.7 TFA and EDT Mean ± standard19.4 ± 0.4 deviation

Results: In both batches, the yield increased by about 5%, indicating asignificant improvement of the peptide cleavage from the solid phase bythe method of the invention.

7.11 Second (Subsequent) Cleavage

After the cleavage, using the comparative cocktail (King's cocktail) orthe cleavage cocktail of the invention, a second (subsequent) cleavage,was performed (see Table 17).

The first cleavage was performed, a filtrate was obtained. TFA was addedto the TFA-wet resin. After 1 h stirring, the resin was filtrated. Thefiltrates were combined and concentrated.

The effect of the second, subsequent cleavage upon lixisenatide yieldwas investigated.

TABLE 29 Influence of a second (subsequent) cleavage of lixisenatideyield. Batch of Modification Subsequent Number of Lixisenatide- ofcleavage cleavage Yield experiment resin (1-44) composition (TFA only)[%] Standard 2B008 26° C., only yes 13.4 ± 1.4 TFA and EDT 70586-0512B008 26° C., only yes 18.5 TFA and EDT 71001-013 2B008 26° C., only yes19.7 TFA and EDT Mean ± 19.1 ± 0.4 standard deviation 70001-018 2B00826° C., only no 17.9 TFA and EDT 70001-019 2B008 26° C., only no 18.1TFA and EDT 70609-078 2B008 26° C., only no 18.1 TFA and EDT 70001-0202B008 26° C., only no 20.1 TFA and EDT Mean ± 18.4 ± 1.1 standarddeviation EDT: 1.2-ethanedithiol.

Results: Subsequent cleavage results in an increase of the yield of onlyabout 0.7%. This increase is associated with a significant increase incosts for starting materials (TFA), and additional efforts to remove theTFA from the peptide preparation. It must be considered that bycombination of the filtrates of the first and second cleavage step, theamount of TFA significantly increases.

It is concluded that in view of the small increase in yield, omission ofthe second cleavage leads to a cost reduction, and handling during theproduction process is facilitated. The amounts of TFA are reduced, sothat removal of TFA is facilitated.

7.12 Analytics

Two batches, 71001-016 (comparative batch, cleavage with King's cocktailaccording to the standard method), and 71001-013 (lixisenatide cleavageaccording to the invention) were prepared.

TABLE 30 analytics Output Content against weight external standardPurity Yield [g] [%] [Fl.-%] [%] 71001-016 3.41 23.0 35.6 14.2(comparative) 71001-013 5.20 20.5 35.9 19.7 (invention)

Results: The batches showed almost identical purity. The content in thebatch produced according to the invention is slightly decreased. In thebatch of the invention, the output weight is increased, resulting in anincreased yield.

7.13 Summary

The cleavage method of the invention has the following advantages:

-   (a) increase of lixisenatide yield by about 5%, resulting in a cost    reduction and an increase of production capacity.-   (b) only two components are present in the cleavage cocktail (in    view of five components in the comparative King's cocktail), thus    analytic quality control is improved and costs are reduced,-   (c) omission of the second cleavage leads to a cost reduction, and    handling during the production process is facilitated. The amounts    of TFA are reduced, so that removal of TFA is facilitated.

The following aspects are also subject of the invention:

-   1. A method for the synthesis of a polypeptide comprising a    pre-determined amino acid sequence, the method comprising coupling    cycles of amino acid building blocks to an amino acid chain,    -   wherein said amino acid building blocks comprise an unprotected        C-terminal carboxyl group and a protected N-terminal amino        group,    -   and wherein said amino acid chain comprises an unprotected        N-terminal amino group,    -   wherein at least one coupling cycle comprises the steps:    -   (a) coupling the amino acid building block C-terminally at the        unprotected N-terminal amino group of the amino acid chain, so        that an amide bond is formed between the amino acid chain and        the amino acid building block,    -   (b) contacting the product obtained in step (a) with a capping        reagent comprising a capping compound, wherein the capping        compound binds to an unprotected N-terminal amino group of the        amino acid chain to which no building block has been coupled in        step (a), and    -   (c) de-protecting the N-terminal amino group of the amino acid        building block.-   2. The method of item 1, wherein the capping compound is selected    from the group consisting of acetic anhydride (CAS 108-24-7),    homologues of acetic anhydride, benzoyl chloride (CAS 98-88-4),    N-(benzyloxycarbonyloxy)succinimide (CAS 13139-17-8), benzyl    chloroformate (CAS 501-53-1), esters of chloroformic acid,    1-acetylimidazole (CAS 2466-76-4), di-tert-butyl dicarbonate (CAS    24424-99-5) and N-(tert-butoxycarbonyloxy)succinimide (CAS    13139-12-3).-   3. The method of item 1 or 2, wherein the capping reagent comprises    0.5-5% v/v of acetic anhydride.-   4. The method of any one of the preceding items, wherein the capping    reagent comprises 1-3% v/v of acetic anhydride.-   5. The method of any one of the preceding items, wherein the capping    reagent comprises 2% v/v of acetic anhydride.-   6. The method of any one of the preceding items, wherein the capping    reagent comprises 0.2-2% v/v of diisopropylethylamine.-   7. The method of any one of the preceding items, wherein the capping    reagent comprises 0.5-2% v/v of diisopropylethylamine.-   8. The method of any one of the preceding items, wherein the capping    reagent comprises 1% v/v of diisopropylethylamine.-   9. The method of any one of the preceding items, wherein the capping    reagent comprises 1% v/v of diisopropylethylamine and 2% v/v of    acetic anhydride.-   10. The method of any one of the preceding items, wherein the    capping reagent comprises DMF.-   11. The method of any one of the preceding items, wherein the    step (b) is performed at a temperature of 15-25° C.-   12. The method of any one of the preceding items, wherein the    step (b) is performed for 5 to 15 min.-   13. The method of any one of the preceding items, wherein the amino    acid building block comprises an α-amino acid.-   14. The method of any one of the preceding items, wherein the amino    acid building block is selected from Ser, Thr, Trp, Lys, Ala, Asn,    Asp, Val, Met, Phe, Ile, Pro, Arg, Glu, Gln, Leu and Gly.-   15. The method of any one of the preceding items, wherein the amino    acid building block is selected from Arg, Glu, Gln, Leu and Gly.-   16. The method of any one of the preceding items, wherein the side    chain of the amino acid building block comprises a protecting group    which is orthogonal to the N-terminal protecting group of the amino    acid building block.-   17. The method of any one of the preceding items, comprising a solid    phase synthesis.-   18. The method of any one of the preceding items, wherein the    N-terminal amino group at the amino acid building block is protected    by a base-labile protecting group.-   19. The method of any one of the preceding items, wherein the    N-terminal amino group at the amino acid building block is protected    by Fmoc.-   20. The method of any one of the preceding items, wherein the    polypeptide is a GLP-1 agonist.-   21. The method of any one of the preceding items, wherein the    polypeptide is selected from GLP-1, analogs and derivatives thereof,    exendin-3, analogs and derivatives thereof, and exendin-4, analogs    and derivatives thereof.-   22. The method of any one of the items 1 to 21, wherein the    polypeptide is selected from exendin-4 and lixisenatide.-   23. The method of any one of the items 1 to 22, wherein the    polypeptide is lixisenatide.-   24. The method of any one of the items 1 to 21, wherein the    polypeptide is selected from albiglutide, dulaglutide and    semaglutide.-   25. The method of in item 1, wherein the polypeptide is    lixisenatide, or exendin-4, wherein after coupling of the amino acid    building block Arg(20), Glu (17), Gln(13), Leu(10) or/and Gly(4),    step (b) is performed for about 10 min with a capping reagent    comprising 2% v/v acetic anhydride and 1% v/v diisopropylethylamine.-   26. Composition, characterized in that it comprises 0.5-5% v/v of    acetic anhydride and 0.2-2% v/v of diisopropylethylamine in DMF.-   27. The composition of item 26, comprising 1% v/v of    diisopropylethylamine and 2% v/v of acetic anhydride.-   28. Use of the composition of item 26 or 27 for acetylation of an    unprotected amino group in polypeptide synthesis.-   29. Use of the composition of item 28, wherein the polypeptide is    lixisenatide.

1. A method for the synthesis of a polypeptide comprising apre-determined amino acid sequence, the method comprising couplingcycles of amino acid building blocks to an amino acid chain, whereinsaid amino acid building blocks comprise an unprotected C-terminalcarboxyl group and a protected N-terminal amino group, and wherein saidamino acid chain comprises an unprotected N-terminal amino group,wherein at least one coupling cycle comprises the steps: (a) couplingthe amino acid building block C-terminally at the unprotected N-terminalamino group of the amino acid chain, so that an amide bond is formedbetween the amino acid chain and the amino acid building block, (b)contacting the product obtained in step (a) with a capping reagentcomprising a capping compound, wherein the capping compound binds to anunprotected N-terminal amino group of the amino acid chain to which nobuilding block has been coupled in step (a), and (c) de-protecting theN-terminal amino group of the amino acid building block.
 2. The methodof claim 1, wherein the capping compound is selected from the groupconsisting of acetic anhydride (CAS 108-24-7), homologues of aceticanhydride, benzoyl chloride (CAS 98-88-4),N-(benzyloxycarbonyloxy)succinimide (CAS 13139-17-8), benzylchloroformate (CAS 501-53-1), esters of chloroformic acid,1-acetylimidazole (CAS 2466-76-4), di-tert-butyl dicarbonate (CAS24424-99-5) and N-(tert-butoxycarbonyloxy)succinimide (CAS 13139-12-3).3. The method of claim 1, wherein the capping reagent comprises 0.5-5%v/v of acetic anhydride.
 4. The method of claim 1, wherein the cappingreagent comprises 0.2-2% v/v of diisopropylethylamine.
 5. The method ofclaim 1, wherein the capping reagent comprises 1% v/v ofdiisopropylethylamine and 2% v/v of acetic anhydride.
 6. The method ofclaim 1, wherein the capping reagent comprises DMF.
 7. The method ofclaim 1, wherein the step (b) is performed at a temperature of 15-25° C.8. The method of claim 1, wherein the step (b) is performed for 5 to 15min.
 9. The method of claim 1, wherein the amino acid building blockcomprises an α-amino acid.
 10. The method of claim 1, wherein the aminoacid building block is selected from Arg, Glu, Gln, Leu and Gly.
 11. Themethod of claim 1, comprising a solid phase synthesis.
 12. The method ofclaim 1, wherein the polypeptide is selected from GLP-1, analogs andderivatives thereof, exendin-3, analogs and derivatives thereof, andexendin-4, analogs and derivatives thereof.
 13. The method of claim 1,wherein the polypeptide is selected from exendin-4, lixisenatide,albiglutide, dulaglutide and semaglutide.
 14. A composition comprising0.5-5% v/v of acetic anhydride and 0.2-2% v/v of diisopropylethylaminein DMF.
 15. Use of the composition of claim 14 for acetylation of anunprotected amino group in polypeptide synthesis.
 16. The method ofclaim 3, where the capping reagent comprises 1-3% v/v of aceticanhydride.
 17. The method of claim 3, where the capping reagentcomprises 2% v/v of acetic anhydride.
 18. The method of claim 4, wherethe capping reagent comprises 0.5-2% v/v of diisopropylethylamine. 19.The method of claim 4, where the capping reagent comprises 1% v/v ofdiisopropylethylamine.
 20. The composition of claim 14, comprising 1%v/v of diisopropylethylamine and 2% v/v of acetic anhydride.